WO2023176881A1 - Combination of multi-specific molecule and immune checkpoint inhibitor - Google Patents

Combination of multi-specific molecule and immune checkpoint inhibitor Download PDF

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WO2023176881A1
WO2023176881A1 PCT/JP2023/010071 JP2023010071W WO2023176881A1 WO 2023176881 A1 WO2023176881 A1 WO 2023176881A1 JP 2023010071 W JP2023010071 W JP 2023010071W WO 2023176881 A1 WO2023176881 A1 WO 2023176881A1
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amino acid
acid sequence
seq
sequence represented
variable region
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PCT/JP2023/010071
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French (fr)
Japanese (ja)
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淳也 市川
谷津 彩香 松井
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第一三共株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates to pharmaceutical compositions containing multispecific molecules, treatment or prevention methods, etc., which are used in combination with immune checkpoint inhibitors and the like.
  • Anticancer drugs include chemotherapy agents such as doxorubicin, carboplatin, taxol, or camptothecin, molecular targeted drugs such as imatinib, crizotinib, dasatinib, and lapatinib, and cancer treatment antibodies such as trastuzumab, bevacizumab, cetuximab, and ramucirumab, depending on the type of cancer.
  • the anticancer drugs and combination therapy to be used are determined depending on the cancer, stage of progression, and treatment status.
  • Immune checkpoint inhibitors which have become standard therapeutic drugs in recent years, are drugs that inhibit the immunosuppressive system and activate antitumor immunity (Non-patent Documents 1 to 3). Immune checkpoint inhibitors include anti-PD-1 antibodies Nivolumab (Patent Document 1), Pembrolizumab (Patent Document 2), and anti-PD-L1 antibodies Atezolizumab (Patent Document 2).
  • Patent Document 3 Durvalumab (Patent Document 4), Avelumab (Patent Document 5), anti-CTLA-4 antibody Ipilimumab (Patent Document 6), Tremelimumab (Patent Document 7) ), Spartalizumab (Patent Document 8), Cemiplimab (Patent Document 9), anti-TIGIT antibodies Tiragolumab (Patent Document 10), Vibostolimab (Patent Document 11), etc. It has been known. There are also cases in which a combination with chemotherapeutic agents has been shown to have a life-prolonging effect (Non-Patent Document 4). Furthermore, cases have been reported in which the combined effects of a cancer antigen and a bispecific antibody targeting CD3 were investigated (Non-patent Documents 5 and 6).
  • Patent Document 12 Although not have.
  • Patent Document 1 International Publication No. 2006/121168 Patent Document 2: International Publication No. 2008/156712 Patent Document 3: International Publication No. 2010/077634 Patent Document 4: International Publication No. 2011/066389 Patent Document 5: International Publication No. 2013/079174 Patent Document 6: International Publication No. 2001/014424 Patent Document 7: International Publication No. 2000/037504 Patent Document 8: International Publication No. 2015/112900 Patent Document 9: International Publication No. 2015/196051 Patent Document 10: International Publication No. 2017/053748 Patent Document 11: International Publication No. 2016/028656 Patent Document 12: International Publication No. 2021/200857
  • Non-patent document 1 Menon S. , et al. , Cancers (2016) 8, 106.
  • Non-Patent Document 2 Pardoll DM. , Nat Rev Cancer (2012) 12, 252-264.
  • Non-Patent Document 3 Wolchok JD. , Cell (2015) 162, 937.
  • Non-patent document 4 Motzer, R. , et al. , New England Journal of Medicine. (2021) 384:1289-300.
  • Non-patent document 5 Deegen P. , et al. , Clinical Cancer Research. (2021) 27:2928-37.
  • Non-patent document 6 Tevernero J. , et al. , Journal of Clinical Oncology. (2017) 35:15_suppl, 3002-3002.
  • One of the objects of the present invention is the combination of a multispecific antibody consisting of an anti-HLA-A2/NY-ESO antibody and an anti-CD3 antibody and other agents, and the use of the antibody or the anti-CD3 antibody in combination with other agents.
  • An object of the present invention is to provide pharmaceutical compositions containing antibodies.
  • Another object of the present invention is to combine a multispecific antibody composed of an anti-HLA-A2/NY-ESO antibody and an anti-CD3 antibody used for the treatment or prevention of cancer with other agents to treat or prevent cancer.
  • An object of the present invention is to provide the antibody or a pharmaceutical composition containing the antibody, which is used for the treatment or prevention of.
  • Another object of the present invention is to administer a multispecific antibody composed of an anti-HLA-A2/NY-ESO antibody and an anti-CD3 antibody in combination with another agent, or to An object of the present invention is to provide a method for treating or preventing cancer by administering a substance.
  • the inventors conducted studies to solve the above problems, and added various anticancer drugs (e.g., The present inventors have discovered that excellent antitumor effects can be obtained by using a combination of chemotherapeutic agents such as carboplatin, paclitaxel, and Nab-paclitaxel, and immune checkpoint inhibitors such as pembrolizumab, and have completed the present invention.
  • chemotherapeutic agents such as carboplatin, paclitaxel, and Nab-paclitaxel
  • immune checkpoint inhibitors such as pembrolizumab
  • the present invention (1) A pharmaceutical composition for the treatment and/or prevention of cancer, comprising the multispecific antibody described in [i] below, which is used in combination with the compound described in [ii] below: [i] Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2, A heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, or a light chain CDRH3 consisting of an amino acid sequence in which the 6th amino acid is N in the amino acid sequence represented by SEQ ID NO: 3, A light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, or a light chain consisting of an amino acid sequence in which the 7th amino acid is W and/or the 8th amino acid is K in the amino acid sequence represented by SEQ ID NO: 4.
  • Light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of Figure 11: SEQ ID NO: 5 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, or A light chain CDRL3 consisting of an amino acid sequence represented by SEQ ID NO: 6 in which the second amino acid is A or S.
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8 or heavy chain CDRH2 consisting of the amino acid sequence in which the third amino acid is N or S in the amino acid sequence represented by SEQ ID NO: 8
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10
  • Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top in Figure 12: SEQ ID NO: 11 in Figure 82) or Arg Asp Asp (fifth sequence from the top in Figure 12: SEQ ID NO: 11 in Figure 82)
  • a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 11) in which the second amino acid is G, Q, N, S or
  • CDRL3 an antibody that binds to CD3 or an antigen-binding fragment thereof, a multispecific antibody, including; [ii] Compounds that inhibit immune checkpoint molecules or chemotherapeutic agents, (2) The pharmaceutical composition according to (1), wherein the multispecific antibody is a bispecific antibody.
  • the antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO binds one or more sets of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following sets (i) to (v).
  • the pharmaceutical composition according to (1) or (2) comprising: (i) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2, Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG.
  • Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, where the seventh amino acid is W
  • a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG.
  • Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2
  • Heavy chain CDRH3 consisting of an amino acid sequence in which the 6th amino acid is N in the amino acid sequence represented by SEQ ID NO: 3
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4
  • a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG.
  • Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, where the second amino acid is A;
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4
  • a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG.
  • a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, in which the second amino acid is S
  • (v) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3
  • Light chain CDRL1 consisting of an amino acid sequence in which the seventh amino acid is W and the eighth amino acid is K in the amino acid sequence represented by SEQ ID NO: 4
  • a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG.
  • the antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO has the amino acid sequence of amino acids 21 to 140 of the amino acid sequence represented by SEQ ID NO: 18, or the amino acid sequence represented by SEQ ID NO: 69 or SEQ ID NO: 70.
  • a heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity, and an amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 18 or SEQ ID NO: 28, or the amino acid sequence represented by SEQ ID NO: 23.
  • a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13 a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ
  • scFv an antibody that binds to HLA/NY-ESO or an antigen-binding fragment thereof, scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 30, scFv comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16, scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20, scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17, scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18, scFv
  • the antibody or antigen-binding fragment thereof that binds to CD3 comprises one or more sets of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following sets (i) to (X), (1) ) to (8): (i) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8, Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9, Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10, Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7
  • Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is N in the amino acid sequence represented by SEQ ID NO: 8
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10
  • Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82)
  • light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7
  • Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10
  • Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82)
  • light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10
  • the light chain CDRL2 consists of an amino acid sequence in which the second amino acid is G, and SEQ ID NO: 12.
  • Light chain CDRL3 consisting of the amino acid sequence shown (v) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8, Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9, Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10, A light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG.
  • Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10
  • a light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG.
  • Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10
  • a light chain CDRL2 consisting of an amino acid sequence in which the second amino acid in the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG.
  • Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
  • Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7
  • Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8
  • Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9
  • Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10
  • a light chain CDRL2 consisting of an amino acid sequence in which the second amino acid is A in the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG.
  • Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12, (iX) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8; Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9, Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10, A light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG.
  • Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12 and (X) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8; Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9, Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10, A light chain CDRL2 consisting of an amino acid sequence in which the second amino acid in the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), and Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12,
  • the pharmaceutical composition according to (9), comprising: (11) An antibody that binds to CD3 or an antigen-binding fragment thereof, A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46, A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47, A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51, A heavy chain variable region consisting of the 2nd to 119th amino acid sequence represented
  • the first polypeptide is represented by the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35.
  • the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38 the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40.
  • the first polypeptide is represented by the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35.
  • the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38 the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40.
  • the first polypeptide includes, from the N-terminus to the C-terminus, an antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO, which is an scFv, an antibody or antigen-binding fragment thereof, which specifically binds to CD3, which is an scFv; Fc region (i) in that order, the second polypeptide consists of an immunoglobulin heavy chain comprising an Fc region (ii); the third polypeptide consists of an immunoglobulin light chain,
  • the second polypeptide and the third polypeptide are associated,
  • the first polypeptide is represented by the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35.
  • the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38 the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40.
  • Amino acid sequence the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence represented by SEQ ID NO: 55 or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, the pharmaceutical composition according to any one of (19) to (22). thing,
  • An antibody that specifically binds to HLA/NY-ESO which is svFv, or an antigen-binding fragment thereof, comprising the heavy chain variable region and constant region CH1 of an antibody that binds to CD3, and immunoglobulin Fc region (i) in that order.
  • first polypeptide a first polypeptide, a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii); and comprising a third polypeptide comprising an antibody light chain that binds to CD3, consisting of a variable region and a constant region
  • first polypeptide and the second polypeptide are associated in Fc region (i) and Fc region (ii)
  • the first polypeptide is associated with a third polypeptide in the variable region and constant region CH1 of the antibody heavy chain.
  • the first polypeptide is the 20th to 724th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 57, the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 60, or the amino acid sequence of SEQ ID NO: 61.
  • the second polypeptide comprises the 20th to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 31.
  • the pharmaceutical composition described, (29) The pharmaceutical composition according to any one of (1) to (28), wherein in the amino acid sequence of one or more polypeptides contained in the pharmaceutical composition, from the carboxyl terminus of the sequence
  • the pharmaceutical composition which has one or two amino acids deleted;
  • the pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is PD-L1; (41) The pharmaceutical composition according to (40), wherein the compound that inhibits an immune checkpoint molecule is atezolizumab, durvalumab, or avelumab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof; (42) The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is CTLA-4; (43) The pharmaceutical composition according to (42), wherein the compound that inhibits an immune checkpoint molecule is ipilimumab, tremelimumab, spartalizumab, or cemiplimab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof; (44) The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is TIGIT; (45) The pharmaceutical composition according to (44), wherein the compound that inhibits an
  • Chemotherapy agents include paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, albumin suspension paclitaxel oxaliplatin, carboplatin, cisplatin, nedaplatin, azacitidine, decitabine, doxorubicin, bleomycin, liposomal doxorubicin, ifosfamide, cyclophosphamide and dacarbazine, the pharmaceutical composition according to any one of (1) to (35) and (48) to (50), (52) The pharmaceutical composition according to any one of (1) to (35) and (48) to (51), wherein the chemotherapeutic agent is paclitaxel or albumin-suspended paclitaxel; (53) The pharmaceutical composition according to any one of (1) to (35) and (48) to (51), wherein the chemotherapeutic agent is paclitaxel or albumin-suspended paclitaxel;
  • composition according to any one of (57) Any one of (53) to (56), wherein the compound that inhibits an immune checkpoint molecule is pembrolizumab, an antigen-binding fragment of pembrolizumab, or a compound containing an antigen-binding fragment thereof, and the multikinase inhibitor is lenvatinib.
  • the pharmaceutical composition according to any one of 48) to (52), (59) A compound or chemotherapeutic agent that inhibits [i] the multispecific antibody and [ii] immune checkpoint molecules according to any one of (1) to (47) for the treatment and/or prevention of cancer.
  • (60) A compound or chemotherapeutic agent that inhibits [i] the multispecific antibody and [ii] immune checkpoint molecules according to any one of (53) to (57) for the treatment and/or prevention of cancer.
  • (61) A compound or chemotherapeutic agent that inhibits [i] the multispecific antibody and [ii] immune checkpoint molecules according to any one of (1) to (47) for the treatment and/or prevention of cancer.
  • (62) [i] Multispecific antibody and [ii] immune checkpoint molecule according to any one of (53) to (57) for preparing a pharmaceutical composition for treating and/or preventing cancer.
  • Figure 1 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Paclitaxel alone, and NYZ-1010 and Paclitaxel in a human T cell transfer model.
  • Figure 1 (2) shows the administration of Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Paclitaxel alone, and NYZ-1010 and Paclitaxel in a human T cell transfer model.
  • FIG. 2 is a diagram showing the tumor volume change rate on Day 31 of each individual with the tumor volume on Day 12 as the baseline in combination administration.
  • Figure 2 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Nab Paclitaxel alone, and NYZ-1010 and NYZ-1010 in a human T cell transfer model.
  • Figure 2 shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Nab Paclitaxel alone, and NYZ-1010 and Nab in a human T cell transfer model. It is a figure showing the tumor volume change rate on Day 38 of each individual with the tumor volume on Day 13 as the baseline in combination administration of Paclitaxel.
  • Figure 3 shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Pembrolizumab alone, and NYZ-1010 and Pembrolizumab combined in a human T cell transfer model.
  • Figure 4 shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYF-0016 alone, anti-mouse PD-1 antibody alone, and NYF-0016 in a human CD3 ⁇ knock-in mouse model.
  • FIG. 2 is a diagram showing the format of antibodies shown in the present invention.
  • scFv A format in which an antibody H chain variable region (VH, described below) and an antibody L chain variable region (VL, described below) (both white) are connected with a linker.
  • VH antibody H chain variable region
  • VL antibody L chain variable region
  • anti-HLA-A2/NY-ESO and CD3 scFv were used for evaluation.
  • Fab A format consisting of VH (white) and antibody H chain constant region (CH1: checkered pattern) and VL (white) and antibody L chain constant region (horizontal line).
  • anti-HLA-A2/NY-ESO Fab etc. were used for evaluation.
  • taFv A format in which two types of scFv (white and upper right diagonal line) are connected by a linker.
  • taFv including anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv were used for evaluation.
  • taFv-heterodimer Fc type Fc with a mutation that forms a heterodimer (hatched line on the upper left) is added to the C-terminal side of taFv (also called the first polypeptide), and another Fc (blacked out: (also referred to as a second polypeptide).
  • taFv-heterodimer Fc containing anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv was used for evaluation.
  • taFv-Fab-heterodimer Fc type This is a format in which Fab is added to the above taFv-heterodimer Fc type.
  • taFv containing anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv, and taFv-Fab-heterodimer Fc using HLA-A2/NY-ESO Fab were used for evaluation.
  • the second polypeptide is a diagram showing the format of antibodies shown in the present invention.
  • (6a) Shows the first polypeptide common to taFv-heterodimer Fc type and taFv-Fab-heterodimer Fc type.
  • the first polypeptide comprises, in order from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA/NY-ESO, an scFv that specifically binds to CD3, and an Fc region (i).
  • (6b) Shows the second polypeptide of taFv-heterodimer Fc type.
  • the second polypeptide comprises a hinge region and an Fc region (ii).
  • FIG. 2 is a diagram showing the format of antibodies included in the present invention.
  • Hybrid type This is a format in which an Fc containing a mutation that forms a heterodimer (hatched and black) is added to the C-terminal side of each of Fab and scFv, and the two Fcs are associated.
  • Dual type This is a format in which an Fc containing a mutation that forms a heterodimer (upper left diagonal line and black) is added to the C-terminal side of each of two different scFvs, and the two Fc heteros are associated.
  • a dual type containing anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv was used for evaluation.
  • scFv-Fab-heterodimer Fc type An Fc with a mutation to form a heterodimer (upper left diagonal line) is added to the C-terminus of the scFv and Fab connected with a linker, and another Fc (blacked out) This is the format in which we met.
  • scFv-Fab-heterodimer Fc type containing anti-CD3 scFv (upper right diagonal line) and anti-HLA-A2/NY-ESO Fab was used for evaluation.
  • FIG. 2 is a diagram showing the format of antibodies shown in the present invention.
  • 8a taFv-heterodimer Fc type: Same as Figure 5(5d). This is a format in which an Fc containing a mutation that forms a heterodimer (shaded in the upper left) is added to the C-terminal side of taFv, and it is heterozygously associated with another Fc (black).
  • taFv-heterodimer Fc type consisting of anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv was used for evaluation.
  • taFv (inverted)-heterodimer Fc type the above taFv-heterodimer Fc type in which the order of the two scFvs is reversed.
  • FIG. 2 is a diagram showing the format of antibodies shown in the present invention.
  • taFv (inverted) - shows the first polypeptide of the heterodimer Fc type.
  • the first polypeptide comprises, in order from the N-terminus to the C-terminus, an scFv that specifically binds to CD3, an scFv that specifically binds to human HLA/NY-ESO, and an Fc region (i).
  • taFv (inverted) - shows a second polypeptide of the heterodimer Fc type.
  • the second polypeptide comprises a hinge region and an Fc region (ii).
  • Amino acid sequence of human CD3 ⁇ It is registered in NCBI/GenPept with accession number: NP_000724 (NM_000724.1). Amino acid sequences of CDRH1-3 and CDRL1-3 contained in NYA-0001 (SEQ ID NO: 1-4 and 6.
  • the fifth is DNN, SEQ ID NO: 5 in Figure 82).
  • Amino acid sequences of heavy chain CDRH1 to CDRH3 and light chain CDRL1 to CDRL3 of C3E-7085 (SEQ ID NO: 7 to 10 and 12; position 11 is RDD; SEQ ID NO: 11 in Figure 82).
  • Amino acid sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO: 13).
  • Amino acid sequence of the light chain variable region of NYA-0001 (SEQ ID NO: 14).
  • Amino acid sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO: 15).
  • Amino acid sequence of the light chain variable region of NYA-0082 (SEQ ID NO: 16).
  • NYA-1163 full-length amino acid sequence (SEQ ID NO: 17). Signal sequence (1-19), NYA-1163 (21-266), FLAG-His tag (267-292). NYA-2023 full-length amino acid sequence (SEQ ID NO: 18). Signal sequence (1-19), NYA-2023 (21-266), FLAG-His tag (267-292). NYA-2027 full-length amino acid sequence (SEQ ID NO: 19). Signal sequence (1-19), NYA-2027 (21-266), FLAG-His tag (267-292). NYA-1143 full-length amino acid sequence (SEQ ID NO: 20). Signal sequence (1-19), NYA-1143 (21-266), FLAG-His tag (267-292). NYA-2143 full-length amino acid sequence (SEQ ID NO: 21).
  • NYF-0022-HC2 full-length amino acid sequence (SEQ ID NO: 34). Signal sequence (1-19), NYA-1163 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). NYF-0023-HC2 full-length amino acid sequence (SEQ ID NO: 35).
  • NYF-0027-HC2 full-length amino acid sequence SEQ ID NO: 36.
  • NYF-0035-HC2 full-length amino acid sequence SEQ ID NO: 37).
  • NYF-0044-HC2 full-length amino acid sequence SEQ ID NO: 38.
  • NYF-0045-HC2 full-length amino acid sequence SEQ ID NO: 39).
  • NYF-0047-HC2 full-length amino acid sequence SEQ ID NO: 40.
  • NYF-0048-HC2 full-length amino acid sequence SEQ ID NO: 41).
  • NYF-0060-HC2 full-length amino acid sequence SEQ ID NO: 42.
  • NYF-0061-HC2 full-length amino acid sequence SEQ ID NO: 43).
  • NYA-0001-Fab-HC1- ⁇ delete full-length amino acid sequence (SEQ ID NO: 44).
  • NYA-0001-LC full-length amino acid sequence SEQ ID NO: 45).
  • C3E-7034 (SEQ ID NO: 46). C3E-7034 (1-267), scFv (2-241), FLAG-His tag (242-267).
  • Amino acid sequence of C3E-7036 (SEQ ID NO: 47). C3E-7036 (1-269), FLAG-His tag (244-269).
  • Amino acid sequence of C3E-7085 (SEQ ID NO: 48). C3E-7085 (1-267), scFv (2-241), FLAG-His tag (242-267). Amino acid sequence of C3E-7088 (SEQ ID NO: 49). C3E-7088 (1-269), FLAG-His tag (244-269).
  • Amino acid sequence of C3E-7093 (SEQ ID NO: 50). C3E-7093 (1-269), FLAG-His tag (244-269). Amino acid sequence of C3E-7078 (SEQ ID NO: 51). C3E-7078 (1-269), FLAG-His tag (244-269).
  • NYF-0014-HC2 full-length amino acid sequence (SEQ ID NO: 52). Signal sequence (1-19), NYA-0001 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). NYF-0082-HC2 full-length amino acid sequence (SEQ ID NO: 53).
  • NYF-0083-HC2 full-length amino acid sequence (SEQ ID NO: 54). Signal sequence (1-19), NYA-2061 (21-266), linker (267-271), C3E-7095 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). NYZ-0082-HC2 full-length amino acid sequence (SEQ ID NO: 55).
  • NYZ-0083-HC2 full-length amino acid sequence (SEQ ID NO: 56). Signal sequence (1-19), NYA-3061 (21-271), linker (272-276), C3E-7097 (277-516), linker (517-518), Hinge (519-533), CH2 (534) -643), CH3 (644-750).
  • NYZ-1010-HC2 full-length amino acid sequence (SEQ ID NO: 57).
  • Figure 72 shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, lenvatinib and Pembrolizumab combined, and NYZ-1010 and NYZ-1010 administered in a human T cell transfer model. It is a figure showing the tumor volume change rate on Day 52 of each individual with the tumor volume on Day 28 as the baseline when lenvatinib and Pembrolizumab were administered in combination.
  • Figure 73 (1) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse PD-1 antibody in a BALB/C strain human CD3 ⁇ knock-in mouse model.
  • Figure 73 (2) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse PD-1 antibody in a BALB/C strain human CD3 ⁇ knock-in mouse model.
  • a diagram showing the estimated tumor volume of each individual after single administration and combined administration of NYZ-1010 and anti-mouse PD-1 antibody, and the estimated tumor volume after CT26 NY-SCT reimplantation in completely regressed mice and naive mice. It is.
  • Figure 74 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Durvalumab alone, and NYZ-1010 and Durvalumab in a human T cell transfer model.
  • Figure 74 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Durvalumab alone, and NYZ-1010 and Durvalumab in a human T cell transfer model.
  • FIG. 75 shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse PD-L1 antibody in a BALB/C strain human CD3 ⁇ knock-in mouse model.
  • Figure 75 shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse CTLA-4 antibody in a BALB/C strain human CD3 ⁇ knock-in mouse model. It is a diagram showing the estimated tumor volume of each individual after single administration and combined administration of NYZ-1010 and anti-mouse CTLA-4 antibody, and the estimated tumor volume after CT26 Mock reimplantation in completely regressed mice and naive mice. .
  • Figure 76 (1) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse CTLA-4 antibody in a BALB/C strain human CD3 ⁇ knock-in mouse model.
  • Figure 76 (2) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse CTLA-4 antibody in a BALB/C strain human CD3 ⁇ knock-in mouse model.
  • FIG. 77 A diagram showing the estimated tumor volume of each individual after single administration and combined administration of NYZ-1010 and anti-mouse CTLA-4 antibody, and the estimated tumor volume after CT26 NY-SCT reimplantation in completely regressed mice and naive mice.
  • Figure 77 (2) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse TIGIT antibody administered alone in the BALB/C strain human CD3 ⁇ knock-in mouse model. , the estimated tumor volume of each individual after combined administration of NYZ-1010 and anti-mouse TIGIT antibody, and the estimated tumor volume after reimplantation of CT26 NY-SCT in completely regressed mice and naive mice.
  • Figure 78 shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Carboplatin alone, and NYZ-1010 and Carboplatin combined in a human T cell transfer model.
  • Figure 79 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, sacituzumab govitecan-hziy administered alone, and NYZ- 1010 and sacituzumab govitecan-hziy when administered in combination.
  • Figure 79 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone administration, sacituzumab govitecan-hziy alone administration, and NYZ-1010 in the human T cell transfer model.
  • FIG. 3 is a diagram showing the rate of change in tumor volume on Day 21 of each individual with the tumor volume on Day 6 as the baseline in the case of combined administration of govitecan-hziy and sacituzumab.
  • Figure 80 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Doxorubicin alone, and NYZ-1010 and Doxorubicin in a human T cell transfer model.
  • Figure 80 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Doxorubicin alone, and NYZ-1010 and Doxorubicin in the human T cell transfer model. It is a figure showing the tumor volume change rate on Day 55 of each individual with the tumor volume on Day 40 as the baseline in combination administration.
  • Figure 81 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Decitabine alone, and NYZ-1010 and Decitabine in a human T cell transfer model.
  • Figure 81 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Decitabine alone, and NYZ-1010 and Decitabine in a human T cell transfer model. It is a figure showing the tumor volume change rate on Day 32 of each individual with the tumor volume on Day 7 as the baseline in combination administration. Sequence list of patent application 2022-041253 filed on March 16, 2022
  • the term “gene” refers to a nucleotide chain containing a base sequence encoding an amino acid of a protein or its complementary chain, such as a nucleotide chain containing a base sequence encoding an amino acid of a protein or its complementary chain. Chains such as polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc. are included in the meaning of "gene”.
  • Such a gene is a single-stranded, double-stranded, or triple-stranded or more nucleotide strand, an aggregate of a DNA strand and an RNA strand, or a mixture of ribonucleotides (RNA) and deoxyribonucleotides (DNA) on a single nucleotide strand.
  • RNA ribonucleotides
  • DNA deoxyribonucleotides
  • gene also included within the meaning of "gene” are genes and double-stranded, triple-stranded or more nucleotide chains containing such nucleotide chains.
  • a base sequence and a nucleotide sequence have the same meaning.
  • polynucleotide In the present invention, "polynucleotide”, “nucleotide chain”, “nucleic acid” and “nucleic acid molecule” have the same meaning, and for example, DNA, RNA, probe, oligonucleotide, primer, etc. are also included in the meaning of "polynucleotide”. .
  • polynucleotides are single-stranded, double-stranded, or polynucleotides consisting of three or more strands, such as an association of a DNA strand and an RNA strand, or a single polynucleotide strand containing ribonucleotides (RNA) and deoxyribonucleotides ( Also included within the meaning of "polynucleotide” are those containing a mixture of polynucleotides (DNA) and double-stranded or three or more stranded aggregates containing such polynucleotide chains.
  • polypeptide In the present invention, “polypeptide”, “peptide” and “protein” have the same meaning.
  • antigen may be used to mean “immunogen.”
  • cells include various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, microorganisms, and the like.
  • antibody is synonymous with immunoglobulin.
  • the term “antibody” is used to mean an immunoglobulin having a constant region and a variable region.
  • the antibody may be a natural immunoglobulin or a partially or completely synthetically produced immunoglobulin.
  • the anti-HLA/NY-ESO antibody of the present invention is included in the "molecule" described below.
  • NY-ESO peptide means a peptide (SLLMWITQC: SEQ ID NO: 1) consisting of NY-ESO-1 and 9 amino acids from the 157th to the 165th amino acids of LAGE-1.
  • HLA-A2/NY-ESO means a complex of NY-ESO peptide and Histocompatibility Leukocyte Antigen-A2 (HLA-A2), and is also written as "HLA/NY-ESO”.
  • anti-HLA-A2/NY-ESO antibody means an antibody that binds to HLA-A2/NY-ESO, in other words, an antibody that recognizes HLA-A2/NY-ESO.
  • anti-HLA-A2/NY-ESO scFv means an scFv that binds to HLA/NY-ESO, in other words, recognizes HLA-A2/NY-ESO.
  • Anti-HLA-A2/NY-ESO antibody” and “anti-HLA-A2/NY-ESO scFv” are also written as “anti-HLA/NY-ESO antibody” and "anti-HLA/NY-ESO scFv,” respectively.
  • the basic four-chain antibody structure is composed of two identical light chains (L chains) and two identical heavy chains (H chains).
  • the light chain is attached to the heavy chain by one covalent disulfide bond.
  • the two heavy chains are linked to each other by one or more disulfide bonds, depending on the heavy chain isotype.
  • Each light and heavy chain has regularly spaced intrachain disulfide bonds.
  • Heavy chains and light chains have a constant region with very high amino acid sequence similarity and a variable region with low amino acid sequence similarity.
  • Light chains have a variable region (VL) at the amino terminus followed by a constant region (CL).
  • the heavy chain has a variable region (VH) at the amino terminus followed by three constant regions (CH1/CH2/CH3).
  • the VL and VH are paired, and the CL is aligned with the first constant region (CH1) of the heavy chain. VL and VH pair to form a single antigen binding site.
  • the constant region of the antibody of the present invention is not particularly limited, human antibodies are preferably used as the antibody of the present invention for treating or preventing human diseases.
  • the heavy chain constant region of human antibodies include C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 4, C ⁇ , C ⁇ , C ⁇ 1, C ⁇ 2, C ⁇ , and the like.
  • the light chain constant region of human antibodies include C ⁇ , C ⁇ , and the like.
  • VH and VL contain complementarity determining regions (CDRs).
  • Fc (also referred to as Fc region) is the carboxyl terminal region of the constant region of the heavy chain, contains CH2 and CH3, and is a dimer.
  • the Fc of the present invention may be a natural sequence Fc or a mutant Fc in which the natural sequence is mutated (referred to as "mutant Fc"), and the multispecific molecule of the present invention and In bispecific molecules, preferred Fc regions are variant Fcs, more preferably a pair of Fcs capable of forming heterodimers.
  • One set of Fc can be a combination of Fc (i) contained in the first polypeptide and Fc (ii) contained in the second polypeptide, which will be described later. dimer formation), it is not limited thereto.
  • mutant Fc examples include modified Fc regions (including heterodimeric Fc regions) contained in heteromultimers with improved stability as disclosed in WO2013/063702, and heterogeneous Fc regions as disclosed in WO1996/27011.
  • Fc containing a CH3 domain contained in a heterodimer which is electrostatically advantageous, in a heterodimer using conformational variation and/or pI (isoelectric point) variation, as disclosed in WO2014/110601.
  • the heterodimeric Fc region include a heterodimeric Fc containing a CH3 domain containing a modification that eliminates or reduces binding to protein A, as disclosed in WO2010/151792. It is not limited to.
  • variable region consists of an extremely variable region called a hypervariable region (HVR) and a relatively constant region called a framework region (FR) separated by this region.
  • HVR hypervariable region
  • FR framework region
  • the variable regions of natural heavy and light chains contain four FRs connected by three hypervariable regions, with the hypervariable regions of each chain being held in close proximity with the hypervariable regions of other chains by the FRs; Contributes to the formation of the antigen-binding site of antibodies.
  • the heavy chain and light chain of an antibody molecule each have three complementarity determining regions (CDRs).
  • the complementarity-determining region also called the hypervariable region, is located within the variable regions of the heavy and light chains of antibodies, and is a region with particularly high variability in primary structure. On the primary structure, each is usually separated into three locations.
  • the complementarity determining region of an antibody is expressed as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence
  • the complementarity determining region of the light chain is expressed as the light chain amino acid sequence.
  • the sequences are expressed as CDRL1, CDRL2, and CDRL3 from the amino terminal side.
  • the positions and lengths of CDRs were determined according to the IMGT definition (Developmental and Comparative Immunology 27 (2003) 55-77).
  • FR is a variable region other than CDR residues.
  • a variable region generally has four FRs: FR1, FR2, FR3, and FR4.
  • the CDRs and FRs included in the heavy chain and light chain are, from the amino terminal to the carboxyl terminal, FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3- FRL4, respectively.
  • CDRs and FRs can also be determined by various definitions well known in the art, for example, in addition to IMGT, Kabat, Chothia, AbM, contact, etc. definitions.
  • the term "antigen-binding fragment of an antibody” refers to a partial fragment of an antibody that is composed of a heavy chain variable region and a light chain variable region and has antigen-binding activity.
  • antigen-binding fragments of antibodies include, but are not limited to, antigen-binding fragments such as Fab, F(ab') 2 , scFv, Fab', Fv, and single-domain antibody (sdAb). It is not something that will be done.
  • antigen-binding fragments of antibodies include those obtained by treating the full-length antibody protein molecules with enzymes such as papain and pepsin, as well as those obtained by recombinant proteins produced in appropriate host cells using recombinant genes. There may be.
  • antibody binding fragment is synonymous with "antibody antigen binding fragment”.
  • the "site" to which an antibody binds refers to a partial peptide or partial conformation on an antigen that the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site.
  • a “mutated antibody” refers to amino acids formed by substitution, deletion, or addition (additions include insertions) of amino acids in the amino acid sequence of the original antibody (hereinafter collectively referred to as "mutations"). It refers to a polypeptide having the sequence and binding to the HLA/NY-ESO of the present invention. The number of mutant amino acids in such a mutant antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40 or 50. Such mutant antibodies are also included in the "antibody” of the present invention.
  • a “multispecific molecule” refers to a molecule containing multiple different epitopes and/or two or more moieties capable of binding to different epitopes on two or more molecules.
  • the molecule is a "multispecific antibody” or a molecule containing a “multispecific antibody”, in which two or more of such parts are antibodies or antigen-binding fragments thereof, respectively, without any particular limitation. It is.
  • a multispecific antibody may include one or more heavy chain variable regions (VH) and/or light chain variable regions (VL), and may contain a complete antibody having two or more different heavy and light chains.
  • Examples of the activities and properties exhibited by the anti-HLA/NY-ESO antibody of the present invention or the antigen-binding fragment thereof, or the molecule of the present invention include biological activity, physicochemical properties (also referred to as physical properties), etc. Specifically, various biological activities such as cytotoxic activity, ADCC activity, and antitumor activity (all described below), binding activity to antigens and epitopes, stability during manufacturing and storage, thermal stability, etc. The physical properties of can be listed.
  • hybridizing under stringent conditions means hybridization is performed at 65°C in a solution containing 5xSSC, and then in an aqueous solution containing 2xSSC-0.1% SDS. 20 minutes at 65°C, 20 minutes at 65°C in an aqueous solution containing 0.5 ⁇ SSC-0.1% SDS, and 65°C in an aqueous solution containing 0.2 ⁇ SSC-0.1% SDS. This means hybridizing for 20 minutes under washing conditions or equivalent conditions.
  • SSC is an aqueous solution of 150mM NaCl-15mM sodium citrate, and nxSSC means n times more concentrated SSC.
  • cytotoxicity refers to any form of pathological change in cells, and includes not only direct trauma but also DNA cleavage, base dimer formation, chromosome breakage, It refers to any structural or functional damage to cells, such as damage to the cell division apparatus or reduction in various enzyme activities.
  • cytotoxic activity means causing the above-mentioned cytotoxicity.
  • antibody-dependent cytotoxic activity refers to “antibody dependent cellular cytotoxicity (ADCC) activity", and refers to the activity of NK cells to damage target cells such as tumor cells via antibodies.
  • ADCC antibody dependent cellular cytotoxicity
  • cytotoxic activity by T cell redirection means causing the above-mentioned cytotoxicity through multispecific molecules including anti-tumor antigen antibodies and anti-HLA/NY-ESO antibodies.
  • the anti-tumor antigen antibody binds to target tumor cells
  • the anti-HLA/NY-ESO antibody binds to T cells, thereby bringing the target tumor cells and T cells closer together and inducing cytotoxicity through T cell activation. It means to do.
  • the molecule can be included in a pharmaceutical composition.
  • HLA/NY-ESO antigen HLA/NY-ESO antigen
  • HLA/NY-ESO HLA/NY-ESO antigen
  • HLA/NY-ESO is a ternary complex of HLA-A2, ⁇ 2-Microglobulin, and NY-ESO peptide.
  • HLA-A2 is a type of HLA allele, and is known as the most frequent allele in Caucasian.
  • HLA forms a tripartite complex of ⁇ 2-microglobulin and a peptide fragment of a self-protein in the endoplasmic reticulum of cells, is presented outside the cell, and is recognized by the TCR (T Cell Receptor) of T cells.
  • NY-ESO peptide (SLLMWITQC: SEQ ID NO: 66) is a peptide consisting of nine amino acids from positions 157 to 165 of NY-ESO-1 and LAGE-1, and is reported to be presented to HLA-A2. .
  • CD3 Antigen in the present invention, "CD3" is used synonymously with CD3 protein.
  • CD3 is expressed on T cells as part of a multimolecular T cell receptor complex, and consists of five types of polypeptides: ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain (with molecular weights of 25,000-28,000, 21,000, 20,000, 16,000, 22,000).
  • CD3 complexes include ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains. These are also called subunits.
  • Anti-CD3 antibodies bind to T cells and induce cytotoxicity through T cell activation. Many anti-CD3 antibodies bind to CD3 ⁇ .
  • the nucleotide sequence of the cDNA encoding human CD3 ⁇ is provided to NCBI/GenBank with accession number: NM_000733 (NM_000733.3), and the amino acid sequence of human CD3 ⁇ is provided to NCBI/GenPept with accession number: NP_000724 (NM_000724.1), respectively. Registered.
  • the nucleotide sequence of cDNA encoding cynomolgus monkey CD3 has been registered in GenBank with accession number: NM_001283615.1.
  • the amino acid sequence of human CD3 ⁇ is shown in FIG.
  • HLA/NY-ESO, CD3 (hereinafter, HLA/NY-ESO and CD3 are collectively referred to as the antigenic proteins) can be used in animal tissues (including body fluids), It can be prepared by purification and isolation from tissue-derived cells or cell cultures, genetic recombination, in vitro translation, chemical synthesis, etc.
  • the cDNA of the antigen protein is obtained by, for example, polymerase chain reaction (hereinafter referred to as "PCR") using a cDNA library of an organ expressing the mRNA of the antigen protein as a template and primers that specifically amplify the cDNA of the antigen protein.
  • PCR polymerase chain reaction
  • the encoding polynucleotide is also included in the cDNA of the antigen protein.
  • a polynucleotide encoding the protein is also included in the cDNA of the antigen protein.
  • the amino acid sequence of the human or rat antigenic protein, or the amino acid sequence from which the signal sequence is removed consists of an amino acid sequence in which one to several amino acids are substituted, deleted, or added, and the antigenic protein is
  • the nucleotide sequence of the antigen protein gene also includes a nucleotide sequence encoding a protein having biological activity equivalent to that of the antigen protein gene.
  • the anti-HLA/NY-ESO antibody and antigen-binding fragment thereof of the present invention recognize HLA/NY-ESO. That is, it binds to HLA/NY-ESO antigen.
  • HLA/NY-ESO is not known to exist in non-human animals such as mice, rats, and cynomolgus monkeys.
  • the anti-CD3 antibody, binding fragment thereof, etc. contained in the multispecific antibody of the present invention recognize, that is, bind to the CD3 antigen.
  • Such anti-CD3 antibodies preferably bind to human CD3, monkey CD3, etc., and more preferably bind to human CD3 and cynomolgus monkey CD3.
  • such preferred anti-CD3 antibodies do not bind to rat and/or mouse CD3.
  • “recognition” or “binding” means binding that is not nonspecific adsorption.
  • a dissociation constant hereinafter referred to as "KD"
  • the KD value of the preferred antibodies of the present invention for HLA/NY-ESO or CD3 is 1 ⁇ 10 ⁇ 5 M or less, 5 ⁇ 10 ⁇ 6 M or less, 2 ⁇ 10 ⁇ 6 M or less, 1 ⁇ 10 ⁇ 6 M
  • the preferred KD values for HLA/NY-ESO are: 5 ⁇ 10 ⁇ 7 M or less, 2 ⁇ 10 ⁇ 7 M or less, 1 ⁇ 10 ⁇ 7 M or less, 5 ⁇ 10 ⁇ 8 M or less, 2 ⁇ 10 ⁇ 8 M or less, 1 ⁇ 10 ⁇ 8 M or less, 5 ⁇ 10 ⁇ 9 M or less, or 2 ⁇ 10 ⁇ 9 M or less, more preferably 1 ⁇ 10 ⁇ 9 M or less.
  • the anti-HLA/NY-ESO scFv of the present invention having excellent antigen binding activity
  • the KD value for HLA/NY-ESO such as NYA-1143, NYA-2044, NYA-2045, NYA-2143 and NYA-1154 is 1 ⁇ 10 ⁇ 9 M or less.
  • the binding between an antigen and an antibody in the present invention can be measured or determined by a biomolecular interaction analysis system such as an SPR method or a BLI method, or an ELISA method or an RIA method (see WO2021/200857).
  • anti-HLA/NY-ESO scFv such as NYA-0001, NYA-1143, NYA-1163, NYA-2023, Examples include NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2061, NYA-2143 and NYA-3061.
  • HLA/NY-ESO is a complex containing HLA-A2 and a 9mer NY-ESO peptide (SLLMWITQC: SEQ ID NO: 66).
  • NY-ESO peptide is a peptide derived from NY-ESO-1 or LAGE-1, which is an intracellular protein and a Cancer-Testis antigen.
  • HLA/NY-ESO is presented on the surface of cancer cells.
  • the anti-HLA/NY-ESO antibody of the present invention and its antigen-binding fragment may be either a monoclonal antibody or a polyclonal antibody.
  • the isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG such as IgG1, IgG2, IgG3, and IgG4, IgM, IgA such as IgA1 and IgA2, IgD, and Ig.
  • the isotype and subclass of a monoclonal antibody can be determined, for example, by the Ochtelony method, ELISA method, RIA method, etc.
  • the monoclonal antibodies of the present invention include antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimerized antibodies (also referred to as "chimeric antibodies"), humanized antibodies, etc., and preferably human antibodies. can be used.
  • the scope of antibodies of the present invention also includes antibody variants (“variant antibodies” described below); for example, the scope of human antibodies also includes human variant antibodies.
  • non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds.
  • mammalian-derived antibodies include rodent-derived antibodies such as mouse antibodies and rat antibodies.
  • antibodies derived from birds include chicken antibodies.
  • chimerized antibodies include, but are not limited to, antibodies formed by combining a variable region derived from a non-human animal antibody and a constant region of a human antibody (human immunoglobulin).
  • Humanized antibodies include those in which CDRs in the variable region of a non-human animal antibody are grafted onto a human antibody (variable region of human immunoglobulin), and in addition to the CDRs, some sequences in the framework region of a non-human animal antibody are also grafted onto a human antibody. Examples include, but are not limited to, those in which one or more amino acids derived from any of these non-human animal antibodies are substituted with human amino acids.
  • Antibodies can be produced by various known methods. As known methods, it can be produced by a method using a hybridoma, a cell immunization method, etc., and can be produced by a genetic recombination method. Additionally, a method for obtaining phage display-derived human antibodies selected from a human antibody library is also known. For example, a phage display method can be used in which the variable region of a human antibody is expressed as an scFv on the surface of a phage and phages that bind to the antigen are selected. By analyzing the genes of phage selected for binding to the antigen, it is possible to determine the DNA sequence encoding the variable region of a human antibody that binds to the antigen.
  • human antibodies can be obtained by constructing an expression vector having the sequence, introducing it into an appropriate host, and expressing it (WO92/01047, WO92/ 20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, Annu. Rev. Immunol (1994) 12, 433-455). It is also possible to use the antibody with high activity obtained in this way as a lead antibody and mutate the gene encoding the lead antibody to produce a variant with higher activity (“mutant antibody” described below). .
  • CDRH1 to CDRH3 contained in the heavy chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention are preferably CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, and CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 2. and heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 3, or the amino acid sequence in which the 6th amino acid in the amino acid sequence represented by sequence number 3 is N (Asn). I can give you some combinations.
  • the NYA-0001 heavy chain variable region consists of the amino acid sequence represented by SEQ ID NO: 13
  • the NYA-0082 heavy chain variable region consists of the amino acid sequence represented by SEQ ID NO: 15
  • the NYA-0082 heavy chain variable region consists of the amino acid sequence represented by SEQ ID NO: 18.
  • CDRL1 to CDRL3 contained in the light chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention preferably has the amino acid sequence represented by SEQ ID NO: 4 or the amino acid sequence represented by SEQ ID NO: 4.
  • CDRL1 consisting of an amino acid sequence in which the seventh amino acid is W (Trp) and/or the eighth amino acid is K (Lys)
  • CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and SEQ ID NO: 6.
  • Examples of CDRL3 combinations include the amino acid sequence represented by SEQ ID NO: 6, or the amino acid sequence in which the second amino acid is A (Ala) or S (Ser).
  • the NYA-0001 light chain variable region consists of the amino acid sequence represented by SEQ ID NO: 14
  • the NYA-0082 light chain variable region consists of the amino acid sequence represented by SEQ ID NO: 16
  • the NYA-0082 light chain variable region consists of the amino acid sequence represented by SEQ ID NO: 20.
  • CDRH1 to CDRH3 contained in the heavy chain and CDRL1 to CDRL3 contained in the light chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention are preferably from the amino acid sequence represented by SEQ ID NO: 1.
  • the amino acid sequence represented by SEQ ID NO: 3 or the amino acid whose 6th amino acid in the amino acid sequence represented by SEQ ID NO: 3 is N (Asn).
  • Heavy chain CDRH3 consisting of the sequence, the amino acid sequence represented by SEQ ID NO: 4, or the 7th amino acid in the amino acid sequence represented by SEQ ID NO: 4 is W (Trp) and/or the 8th amino acid is K (Lys) or , a combination of CDRL3 consisting of an amino acid sequence represented by SEQ ID NO: 6 in which the second amino acid is A (Ala) or S (Ser). More preferably, NYA-0001 heavy chain variable region and light chain variable region consisting of the amino acid sequences represented by SEQ ID NO: 13 and 14, respectively, and amino acid numbers 21-140 and 156- of the amino acid sequence represented by SEQ ID NO: 20.
  • NYA-1143 heavy chain variable region and light chain variable region consisting of 266, NYA-1163 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 17, NYA-2023 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 18, amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 19, and NYA-2027 heavy chain variable region and light chain variable region consisting of amino acid numbers 156 to 266, NYA-2035 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 22 NYA-2044 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 24, and amino acid numbers 21 to 21 of the amino acid sequence represented by SEQ ID NO: 25.
  • NYA-2045 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 26; Chain variable region, NYA-2048 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 27, amino acid number of the amino acid sequence represented by SEQ ID NO: 28
  • NYA-2060 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266, NYA-2061 heavy chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 29 and light chain variable region, or CDRH1 to CDRH3 and Combinations of CDRL1 to CDRL3 can be listed.
  • a combination of CDRH1 to CDRH3 and CDRL1 to CDRL3 respectively contained in the NYA-3061 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 161 to 271 of the amino acid sequence represented by SEQ ID NO: 55, And, list the combinations of CDRH1 to CDRH3 and CDRL1 to CDRL3 contained in the NYA-1154 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 71. Can be done.
  • the heavy chain variable region of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned heavy chain CDRs or a combination thereof, and more preferably, NYA-0001 heavy chain variable region, NYA-0082 heavy chain variable region, NYA-1143 heavy chain variable region, NYA-1163 heavy chain variable region, NYA-2023 heavy chain variable region, NYA-2027 heavy chain variable region, NYA- 2035 heavy chain variable region, NYA-2044 heavy chain variable region, NYA-2045 heavy chain variable region, NYA-2047 heavy chain variable region, NYA-2048 heavy chain variable region, NYA-2060 heavy chain variable region, NYA-2061 heavy chain variable region Examples include the chain variable region, NYA-2143 heavy chain variable region, NYA-3061 heavy chain variable region, and NYA-1154 heavy chain variable region.
  • the amino acid sequence of each heavy chain variable region is as described above.
  • the light chain variable region of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned light chain CDRs or a combination thereof, and more preferably, NYA-0001 light chain variable region, NYA-0082 light chain variable region, NYA-1143 light chain variable region, NYA-1163 light chain variable region, NYA-2023 light chain variable region, NYA-2027 light chain variable region, NYA- 2035 light chain variable region, NYA-2044 light chain variable region, NYA-2045 light chain variable region, NYA-2047 light chain variable region, NYA-2048 light chain variable region, NYA-2060 light chain variable region, NYA-2061 light chain variable region Examples include the chain variable region, NYA-2143 light chain variable region, NYA-3061 light chain variable region, and NYA-1154 light chain variable region.
  • the amino acid sequence of each light chain variable region is as described above.
  • the heavy chain variable region and light chain variable region of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned heavy chain and light chain CDRs or a combination thereof.
  • NYA-0001 heavy chain variable region and light chain variable region NYA-0082 heavy chain variable region and light chain variable region, NYA-1143 heavy chain variable region and light chain variable region, NYA- 1163 heavy chain variable region and light chain variable region, NYA-2023 heavy chain variable region and light chain variable region, NYA-2027 heavy chain variable region and light chain variable region, NYA-2035 heavy chain variable region and light chain variable region, NYA-2044 heavy chain variable region and light chain variable region, NYA-2045 heavy chain variable region and light chain variable region, NYA-2047 heavy chain variable region and light chain variable region, NYA-2048 heavy chain variable region and light chain variable region region, NYA-2060 heavy chain variable region and light chain variable region, NYA-2061 heavy chain variable region and light chain variable region, NYA-2143 heavy chain variable region and light chain variable region combination, NYA-3061 heavy chain variable region and light chain variable regions, and combinations of NYA-1154 heavy chain variable regions and light chain variable regions.
  • Preferred examples of the heavy chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned preferred or more preferred heavy chain variable regions, and more preferably, NYA-0001 heavy chain, NYA-0082 heavy chain, NYA-1143 heavy chain, NYA-1163 heavy chain, NYA-2023 heavy chain, NYA-2027 heavy chain, NYA-2035 heavy chain, NYA-2044 heavy chain, NYA- Examples include 2045 heavy chain, NYA-2047 heavy chain, NYA-2048 heavy chain, NYA-2060 heavy chain, NYA-2061 heavy chain, NYA-2143 heavy chain, NYA-3061 heavy chain, and NYA-1154 heavy chain. be able to.
  • Preferred examples of the light chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned preferred or more preferred light chain variable regions, and more preferably, NYA-0001 light chain, NYA-0082 light chain, NYA-1143 light chain, NYA-1163 light chain, NYA-2023 light chain, NYA-2027 light chain, NYA-2035 light chain, NYA-2044 light chain, NYA- Examples include 2045 light chain, NYA-2047 light chain, NYA-2048 light chain, NYA-2060 light chain, NYA-2061 light chain, NYA-2143 light chain, NYA-3061 light chain, and NYA-1154 light chain. be able to.
  • Preferred examples of the heavy chain and light chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned preferred or more preferred heavy chain variable region and light chain variable region, respectively. More preferably, NYA-0001, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA Examples include heavy and light chain combinations of -2060, NYA-2061, NYA-2143, NYA-3061, and NYA-1154.
  • An antigen-binding fragment of an antibody means a fragment or a modified product thereof that retains at least antigen-binding properties among the functions of the antibody.
  • Functions of such antibodies generally include antigen-binding activity, antigen activity-regulating activity, antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, and the like.
  • the functions of the antibodies of the present invention and multispecific molecules containing the antibodies of the present invention include, for example, T cell redirection, T cell activation, and cancer cell activation by activating T cells. Cytotoxic activity can be mentioned.
  • Antigen-binding fragments of antibodies are not particularly limited as long as they retain at least antigen-binding activity of the antibody, but examples include Fab, Fab', F(ab') 2 , Examples include, but are not limited to, Fv, single chain Fv (scFv) in which heavy chain and light chain Fv are linked with a suitable linker, and single domain antibody (sdAb). Molecules containing portions other than the antigen-binding fragment of the antibody of the present invention, such as scFv carrying a linker portion, are also included within the meaning of the antigen-binding fragment of the antibody of the present invention.
  • a molecule in which one or more amino acids at the amino terminus and/or carboxyl terminus of an antibody protein are deleted, and which retains at least a part of the functions of the antibody, is also an antigen-binding fragment of an antibody.
  • an antigen-binding fragment of an antibody included in the meaning of Modified forms of such antigen-binding fragments of antibodies are also included in the antibody or antigen-binding fragment thereof of the present invention, or modified forms thereof (described below).
  • scFv is obtained by linking the heavy chain variable region and light chain variable region of an antibody with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, edited by Rosenburg and Moore, Spring ger Verlag, New York, 269- 315 (1994), Nature Biotechnology (2005), 23, 1126-1136). Additionally, tandem scFvs produced by linking two scFvs with a polypeptide linker can also be used as bispecific molecules. Furthermore, triabodies and the like composed of three or more scFv can also be used as multispecific molecules.
  • the HLA/NY-ESO-specific scFv (also called “anti-HLA/NY-ESO scFv”) preferably includes the above-mentioned CDRH1 to CDRH3 and CDRL1 to CDRL3, and more preferably the above-mentioned heavy chain variable region.
  • NYA-0001 amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 30
  • NYA-0082 amino acid sequence represented by SEQ ID NO: 15
  • including the amino acid sequence represented by SEQ ID NO: 16 NYA-1143 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 20), NYA-1163 (amino acid numbers of the amino acid sequence represented by SEQ ID NO: 17) 21-266), NYA-2023 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 18), NYA-2027 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 19), NYA-2035 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 22), NYA-2044 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 24), NYA-2045 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO:
  • NYA-3061 amino acid numbers 21 to 271 of the amino acid sequence represented by SEQ ID NO: 55
  • NYA-1154 amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 71
  • a preferred embodiment of the anti-HLA/NY-ESO scFv includes one in which a FLAG-His tag is fused to the carboxyl terminal side (also simply referred to as a "tag adduct").
  • Suitable tag adducts include NYA-0001 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 30), NYA-1143 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 20), and NYA-1163 tag adduct.
  • NYA-2061 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 29), NYA-2143 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 21), NYA-3061FLAG-His tag adduct (amino acid sequence formed by adding amino acid numbers 267 to 292 of SEQ ID NO: 22 to the carboxyl terminal of amino acid numbers 21 to 271 of SEQ ID NO: 56), and NYA-1154 tag adduct (amino acid numbers 21 to 292 of SEQ ID NO: 71) ) can be given.
  • NYA-2023 and its tagged product NYA-2047 and its tagged product
  • NYA-2048 and its tagged product NYA-2060 and its tagged product
  • NYA-2061 and its tagged product is more suitable as an Fc-added bispecific molecule because it has excellent biological activity, physical properties, etc.
  • a signal peptide when anti-HLA/NY-ESO scFv and its tagged product are expressed in host cells, a signal peptide can be added to the amino terminus.
  • the amino acid sequences of the anti-HLA/NY-ESO scFv tag adduct with a signal peptide include the amino acids of SEQ ID NO: 30, 20, 17-19, 22, 24, 25, 26-29, 21, and SEQ ID NO: 56.
  • An amino acid sequence in which a FLAG-His tag (amino acid numbers 267 to 292 of SEQ ID NO: 22) is added to the carboxyl terminus of numbers 21 to 271 can be mentioned.
  • scFv is a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105) in which the variable region of an antibody is expressed on the surface of a phage as a single chain antibody (scFv) and phages that bind to the antigen are selected. -1116). By analyzing the genes of phage selected for binding to the antigen, it is possible to determine the DNA sequence encoding the variable region of a human antibody that binds to the antigen.
  • human antibodies can be obtained by constructing an expression vector containing the sequence, introducing it into an appropriate host, and expressing it (WO92/01047, WO92/ 20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, Annu.Rev.Immunol (1994) 12, p.433-455, Nature Biotechnology (2005) ) 23(9), p. 1105-1116).
  • the antibody of the present invention may have a single heavy chain variable region and no light chain sequence.
  • Such antibodies are called single domain antibodies (sdAbs) or nanobodies, and it has been reported that they retain antigen-binding ability (Muyldemans S. et. al. , Protein Eng., (1994) 7(9), 1129-35, Hamers-Casterman C. et. al., Nature (1993) 363(6428), 446-448).
  • sdAbs single domain antibodies
  • These antibodies are also included within the meaning of antigen-binding fragments of antibodies in the present invention.
  • the present invention also includes a single chain immunoglobulin in which the full-length sequences of the heavy chain and light chain of an antibody are linked using an appropriate linker (Lee, HS, et. al. , Molecular Immunology (1999) 36, 61-71; Shirrmann, T. et. al., mAbs (2010), 2(1), 1-4).
  • an appropriate linker Lee, HS, et. al. , Molecular Immunology (1999) 36, 61-71; Shirrmann, T. et. al., mAbs (2010), 2(1), 1-4.
  • the anti-HLA/NY-ESO antibody of the invention may be a single chain immunoglobulin.
  • the heavy chain variable region and the light chain variable region may be disulfide bonded.
  • the anti-HLA/NY-ESO antibody of the present invention may be an antibody composed of parts derived from a plurality of different antibodies, as long as it binds to HLA/NY-ESO. Those in which the chain and/or light chain are exchanged, those in which the entire length of the heavy chain and/or light chain are exchanged, those in which only the variable region or only the constant region are exchanged, those in which only all or part of the CDRs are exchanged, etc. can be mentioned.
  • the heavy chain variable region and light chain variable region of the chimerized antibody may be derived from different anti-HLA/NY-ESO antibodies of the invention.
  • Heavy chain CDR1 to heavy chain CDR3 and light chain CDR1 to light chain CDR3 in the heavy chain and light chain variable regions of the humanized antibody are derived from two or more anti-HLA/NY-ESO antibodies of the present invention. It's okay. Heavy chain CDR1 to heavy chain CDR3 and light chain CDR1 to light chain CDR3 in the heavy chain and light chain variable regions of human antibodies are the CDRs possessed by two or more anti-HLA/NY-ESO antibodies of the present invention. It may be a combination.
  • the anti-HLA/NY-ESO antibody of the present invention is hybridized under stringent conditions with a complementary strand of a polynucleotide containing a nucleotide sequence encoding the amino acid sequence contained in the anti-HLA/NY-ESO antibody of the present invention. Also included are antibodies that contain the amino acid sequence encoded by the nucleotide sequence contained in the polynucleotide and that bind to HLA/NY-ESO.
  • amino acid sequence contained in the heavy chain variable region of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention preferably the amino acid sequence of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 18, The amino acid sequence represented by No. 69 or SEQ ID No. 70, the amino acid sequence represented by SEQ ID No. 39, or the amino acid number 21 to 140 of the amino acid sequence represented by SEQ ID No. 57, SEQ ID No. 60, or SEQ ID No. 61.
  • amino acid sequence preferably the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 18, the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28
  • Amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to and an antibody or an antigen-binding fragment thereof that binds to HLA/NY-ESO.
  • the position and length of the light chain variable region is determined by the IMGT definition when it is determined using a definition different from IMGT (e.g. Kabat, Chothia, AbM, contact, etc.) than when it is determined by the IMGT definition.
  • the carboxyl terminus of the light chain variable region amino acid sequence may further include one or more amino acids, such as arginine or glycine.
  • Antibodies or binding fragments thereof having such light chain variable regions are also included in the antibodies or binding fragments thereof of the present invention.
  • the antibodies and the like of the present invention have the ability to bind to HLA/NY-ESO, particularly human and/or cynomolgus monkey HLA/NY-ESO, by introducing mutations into the binding fragment of the anti-HLA/NY-ESO antibody of the present invention. It may be an optimized one. Specific methods for introducing mutations include random mutagenesis using error-prone PCR, site-specific amino acid mutagenesis using NNK libraries, site-specific mutagenesis using structural information, and combinations thereof. can be mentioned.
  • mutant antibody variant of anti-HLA/NY-ESO antibody
  • the mutant antibody of the anti-HLA/NY-ESO antibody of the present invention preferably has a reduced sensitivity to protein degradation or oxidation, maintains, improves, or suppresses a decline or change in biological activity or function, and improves antigen binding ability. or adjustment, or imparting physicochemical properties or functional properties, etc. It is known that changes in specific amino acid side chains on the surface of proteins can change the function and activity of the protein. Examples of such changes include deamidation of asparagine side chains and deamidation of aspartate side chains. Includes isomerization, etc. Mutant antibodies of the present invention also include antibodies in which other amino acids are substituted to prevent such changes in amino acid side chains.
  • mutant antibody of the present invention includes an antibody having an amino acid sequence resulting from conservative amino acid substitutions in the amino acid sequence of the antibody.
  • Conservative amino acid substitutions are those that occur within a group of amino acids that are related in their amino acid side chains.
  • Mutant antibodies that have an amino acid sequence in which conservative amino acid substitutions and/or other mutations have been made in the amino acid sequence of the antibody of the present invention such as NYA-2023, and that bind to HLA/NY-ESO, and their antigen binding Fragments, molecules containing them, etc.; Chimerized antibodies, humanized antibodies, human antibodies, antigen-binding fragments thereof, molecules containing them, etc., which contain the CDRs and which bind to HLA/NY-ESO, are also included in the anti-HLA/NY-ESO antibodies of the present invention. , an antigen-binding fragment thereof, a variant thereof (a mutant antibody or an antigen-binding fragment thereof), or a molecule of the present invention.
  • binding fragment an antigen-binding fragment (hereinafter simply referred to as "binding fragment") of the anti-HLA/NY-ESO antibody of the present invention.
  • the binding fragments of anti-HLA/NY-ESO antibodies of the present invention include binding fragments of chimerized antibodies, humanized antibodies, or human antibodies.
  • the antibody binding fragment means a fragment or a modified version thereof that retains at least antigen-binding properties among the functions of the antibody.
  • the functions of such antibodies generally include antigen-binding activity, activity to modulate antigen activity (for example, agonist activity), activity to internalize the antigen into cells, and interaction with substances that interact with the antigen. Examples include activities that inhibit or promote.
  • the antibody binding fragment is not particularly limited as long as it is a fragment of the antibody that retains at least the antigen binding activity of the antibody.
  • Such antibody binding fragments include, for example, Fab, Fab', F(ab') 2 single chain Fab (scFab) in which the carboxyl terminus of Fab light chain and the amino terminus of Fab heavy chain are linked with an appropriate linker. ), Fv, single chain Fv (scFv) in which heavy and light chain Fv are linked with a suitable linker, single domain antibody (sdAb) with a single heavy chain variable region and no light chain sequence. ) or also called nanobody.
  • Modified products and complexes of anti-HLA/NY-ESO antibodies or binding fragments thereof The present invention provides modified products of antibodies or binding fragments thereof.
  • a modified antibody of the present invention or a binding fragment thereof means an antibody of the present invention or a binding fragment thereof that has been chemically or biologically modified.
  • Chemical modifications include attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like.
  • Biological modifications include post-translational modifications such as N- or O-linked glycosylation, processing of amino- or carboxyl-terminal regions, deamidation, aspartic acid isomerization, methionine oxidized), and those with a methionine residue added to the amino terminus by expression using prokaryotic host cells.
  • post-translational modifications such as N- or O-linked glycosylation, processing of amino- or carboxyl-terminal regions, deamidation, aspartic acid isomerization, methionine oxidized), and those with a methionine residue added to the amino terminus by expression using prokaryotic host cells.
  • those labeled to enable detection or isolation of the antibody or antigen of the present invention such as enzyme labels, fluorescent labels, and affinity labels, are also included in the meaning of such modifications.
  • Such modified antibodies of the present invention or binding fragments thereof can be used to improve the stability and retention in blood of the original antibodies of the present invention or binding fragments thereof, to reduce antigenicity,
  • Examples of the chemical moiety included in the chemically modified product include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, and polyvinyl alcohol.
  • PEG polyethylene glycol
  • ethylene glycol/propylene glycol copolymer ethylene glycol/propylene glycol copolymer
  • carboxymethyl cellulose cellulose
  • dextran dextran
  • polyvinyl alcohol polyvinyl alcohol
  • Bioly modified products include those modified by enzyme treatment or cell treatment, fusions with other peptides such as tags added through genetic recombination, and endogenous or exogenous glycosylation enzymes. Examples include those prepared using cells expressing the expression as a host.
  • Such modifications may be made at any position or at a desired position in the antibody or binding fragment thereof, and the same or two or more different modifications may be made at one or more positions.
  • the present invention also includes antibodies with such deletions or modifications.
  • a deletion product in which one or two amino acids are deleted at the carboxyl terminal of the heavy chain Journal of Chromatography A; 705; 129-134 (1995)
  • glutamine or glutamic acid at the amino terminal of the heavy chain or light chain of the antibody Antibodies whose residues have been modified by pyroglutamylation (International Patent Publication No. WO2013/147153) can be mentioned (these are collectively referred to as "deletion bodies").
  • the carboxyl terminal deletions of the heavy chain and light chain of the antibody according to the present invention are not limited to the above types, as long as the antigen binding ability and effector function are maintained.
  • the two or more chains (e.g. heavy chains) are heavy chains selected from the group consisting of full-length and deletion forms as described above. It may be one of these or a combination of two of them.
  • the amount ratio or molecular number ratio of each deletion product may be influenced by the type and culture conditions of cultured mammalian cells producing the antibody according to the present invention, the main components of the antibody according to the present invention are two heavy chains.
  • One example is a case where one amino acid residue at the carboxyl terminus is deleted in both.
  • the antibody of the present invention or its antigen-binding fragment (such as those included in the multispecific antibody of the present invention) has one to several amino acids derived from the expression vector and/or signal sequence, etc. at the amino terminal and/or Modifications of the modified antibody of the present invention or antigen-binding fragment thereof, as long as the desired antigen-binding activity is maintained even if the modified antibody is added to the carboxyl terminus (and a part or all of it is modified as described above).
  • multispecific antibodies that are within the scope of the human body and include modifications of such antibodies or antigen-binding fragments.
  • antibody or binding fragment thereof also includes “modified antibody or antigen-binding fragment thereof". Furthermore, the “antibody or antigen-binding fragment thereof” included in the multispecific antibody of the present invention includes within its meaning such "modified form of the antibody or antigen-binding fragment thereof.”
  • the present invention also includes a complex (Immunoconjugate) in which the above-mentioned antibody and other molecules are connected with a linker.
  • a complex Immunoconjugate
  • An example of an antibody-drug conjugate in which the antibody is bound to a radioactive substance or a compound (drug) having pharmacological action is ADC (Antibody-Drug Conjugate) ((Methods Mol Biol. (2013)). 1045:1-27; Nature Biotechnology (2005) 23, p. 1137-1146).
  • the present invention also includes complexes in which these antibodies are linked to other functional polypeptides.
  • An example of such an antibody-peptide complex is a complex in which the antibody is combined with an albumin-binding polypeptide (Protein Eng Des Sel. (2012) (2): 81-8).
  • the above-mentioned modified antibodies, antibodies with regulated glycosylation, and complexes are included in the antibody of the present invention, and binding fragments of the above-mentioned modified antibodies, antibodies with regulated glycosylation, and conjugates are included in the antibody of the present invention. , are included in the binding fragments of the antibodies of the invention. 4.
  • the antibody of the present invention can be produced by, for example, inserting a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region into an expression vector, transforming a host cell for expression with the vector, and By culturing cells, recombinant antibodies can be produced in the cells.
  • the DNA encoding the antibody is obtained by linking the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region
  • the DNA encoding the heavy chain is obtained by linking the DNA encoding the heavy chain variable region and the DNA encoding the light chain constant region.
  • DNA encoding the light chain is obtained by ligating the DNA encoding the chain constant region.
  • the anti-HLA/NY-ESO antibody of the present invention can be obtained by inserting the above-mentioned heavy chain-encoding DNA and light chain-encoding DNA into an expression vector, transforming host cells with the vector, and culturing the host cells. It can be produced by At this time, the DNA encoding the heavy chain and the DNA encoding the light chain described above may be introduced into the same expression vector, and the vector may be used to transform host cells, or the DNA encoding the heavy chain and the DNA encoding the light chain may be introduced into the same expression vector.
  • the DNA encoding the strands may be inserted into separate vectors and the two vectors used to transform host cells.
  • the DNA encoding the heavy chain variable region and the light chain variable region may be introduced into a vector into which DNA encoding the heavy chain constant region and DNA encoding the light chain constant region have been introduced in advance.
  • the vector may contain a DNA encoding a signal peptide that promotes secretion of the antibody from the host cell.
  • the DNA encoding the signal peptide and the DNA encoding the antibody are linked in frame. put. By removing the signal peptide after the antibody has been produced, the antibody can be obtained as a mature protein.
  • DNA encoding the heavy chain variable region DNA encoding the light chain variable region, DNA encoding the heavy chain variable region and DNA encoding the heavy chain constant region, and DNA encoding the light chain variable region.
  • a DNA obtained by linking a DNA and a DNA encoding a light chain constant region may be functionally linked to elements such as a promoter, an enhancer, and a polyadenylation signal. Functionally connected here means that elements are connected so that they perform their functions.
  • the expression vector is not particularly limited as long as it can be replicated in a host such as animal cells, bacteria, yeast, etc., and includes, for example, known plasmids, phages, and the like.
  • Vectors used for constructing expression vectors include, for example, pcDNA (trademark) (ThermoFissher SCIENTIFIC), Flexi (registered trademark) vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2. (manufactured by Pharmacia), pMAM-neo (manufactured by Clontech), and the like.
  • prokaryotic cells such as Escherichia coli and Bacillus subtilis
  • eukaryotic cells such as yeast and animal cells
  • eukaryotic cells are preferably used.
  • animal cells HEK293 cells, which are human embryonic kidney cell lines, Chinese hamster ovary (CHO) cells, etc.
  • the expression vector may be introduced into a host cell by a known method and the host cell may be transformed. Examples include electroporation method, calcium phosphate precipitation method, DEAE-dextran transfection method, and the like.
  • the produced antibodies can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatography, filters, ultrafiltration, salting out, dialysis, etc. may be appropriately selected and combined.
  • Multispecific antibodies that bind to HLA/NY-ESO Multispecific antibodies of the invention include anti-HLA/NY-ESO antibodies of the invention or antigen-binding fragments thereof.
  • the multispecific antibody of the present invention preferably has two or more antigen binding sites. That is, it is a multispecific antibody capable of binding to two or more different epitopes on one molecule or to different epitopes on two or more molecules, and encompasses a plurality of different antigen-binding fragments.
  • Such multispecific antibodies include IgG type multispecific antibodies, multispecific antibodies having two or more types of variable regions, such as tandem scFv (taFv), single chain diabodies, diabodies and triabodies. including, but not limited to, antibody fragments that are covalently or non-covalently linked. Multispecific antibodies may include Fc.
  • the multispecific antibody of the present invention may contain, in addition to the anti-HLA/NY-ESO antibody of the present invention or an antigen-binding fragment thereof, one or more additional antibodies or antigen-binding fragments of the antibody.
  • Additional antigen-binding fragments of antibodies can include, for example, Fab, F(ab)', Fv, scFv, sdAb.
  • a preferred multispecific antibody of the present invention further comprises an anti-CD3 antibody or antigen-binding fragment thereof, and also specifically binds to CD3.
  • the anti-CD3 antibody or antigen-binding fragment thereof contained in the multispecific antibody of the present invention is not particularly limited as long as it is an antibody or a binding fragment thereof that binds to human CD3, but is preferably a non-human antibody such as a cynomolgus monkey. It also binds to primate CD3. More preferred anti-CD3 antibodies or antigen-binding fragments thereof include heavy chain variable region CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, heavy chain variable region CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8, and heavy chain variable region CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8.
  • Heavy chain variable region CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9; light chain variable region CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10; ) and an antibody or an antigen-binding fragment thereof comprising a light chain variable region CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
  • a more preferable antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 is a C3E-7034 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 46; C3E-7036 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 47, and C3E- consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 48.
  • 7085 heavy chain variable region consisting of the amino acid sequence of amino acids 2 to 119 of the amino acid sequence represented by SEQ ID NO: 49, C3E-7088 heavy chain variable region consisting of the amino acid sequence of amino acids 2 to 119 of the amino acid sequence represented by SEQ ID NO: 140.
  • C3E-7093 heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 54
  • C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acids 272 to 389 of the amino acid sequence represented by SEQ ID NO: 54
  • the C3E-7096 heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 55.
  • An antibody comprising a C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 277 to 394, and a C3E-7097 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 277 to 394 of the amino acid sequence represented by SEQ ID NO: 56. or an antigen-binding fragment thereof.
  • a more preferred antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 is C3E-7034 light chain variable consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO: 46.
  • C3E-7036 light chain variable region consisting of the amino acid sequence of amino acids 135 to 241 of the amino acid sequence represented by SEQ ID NO: 47, consisting of the amino acid sequence of amino acids 135 to 241 of the amino acid sequence represented by SEQ ID NO: 48
  • C3E-7085 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO: 49
  • C3E-7088 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO: 50
  • C3E-7093 light chain variable region consisting of the amino acid sequence of 243
  • C3E-7096 light chain variable region consisting of the amino acid sequence of amino acids 405 to 511 of the amino acid sequence represented by SEQ ID NO: 54, amino acid represented by SEQ ID NO: 55.
  • Examples include antibodies or antigen-binding fragments thereof.
  • more preferable antibodies or antigen-binding fragments thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 include the C3E-7034 heavy chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 represented by SEQ ID NO: 46; Combination of light chain variable regions, combination of C3E-7036 heavy chain variable regions and light chain variable regions consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 47, amino acid number 2 represented by SEQ ID NO: 51
  • An example is an antibody or an antigen-binding fragment thereof comprising a combination of the C3E-7097 heavy chain variable region and light chain variable region consisting of 410-516.
  • antibodies or antigen-binding fragments thereof containing the CDRH1 to CDRH3 and CDRL1 to CDRL3 include C3E-7034scFv, which consists of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence represented by SEQ ID NO: 46; C3E-7036scFv consisting of the amino acid sequence of amino acid numbers 2 to 241 of the amino acid sequence represented by SEQ ID NO. C3E-7085scFv consists of the amino acid sequence of amino acid numbers 2 to 241 of the amino acid sequence represented by SEQ ID NO. 49, C3E-7088scFv consists of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence of SEQ ID NO.
  • CD3-specific scFv also referred to as "anti-CD3 scFv”
  • FLAG-His tag added to the carboxyl terminal side
  • Suitable tag adducts include C3E-7034 (SEQ ID NO: 46), C3E-7036 (SEQ ID NO: 47), C3E-7085 (SEQ ID NO: 48), C3E-7088 (SEQ ID NO: 49) and C3E-7093 (SEQ ID NO: 49). 50), and more preferably C3E-7085.
  • a preferred example of the multispecific molecule of the present invention is a bispecific molecule.
  • Bispecific means capable of binding to two different epitopes on the same molecule or to different epitopes on two molecules; an antibody or Includes antigen-binding fragments.
  • the bispecific molecules of the invention bind to HLA/NY-ESO and further bind to CD3.
  • bispecific molecule of the present invention examples include those having the following structure (format).
  • a dual scFv type bispecific molecule two types of scFv that bind to different epitopes are each linked to one of the dimeric Fcs with a linker or directly linked without a linker.
  • two types of scFv that bind to different epitopes are each linked to CH and CL with a linker, and each is further linked to one of the dimeric Fcs with a linker.
  • the bispecific molecule is in a format in which two different scFvs have heterozygous Fcs containing mutations that form heterodimers downstream of each.
  • a dual scFv type bispecific molecule is called a dual type bispecific molecule or simply a dual type ( Figure 7 (7b)).
  • a dual-type bispecific molecule consisting of anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv may be used.
  • a bispecific molecule of the invention a Fab and scFv that bind to different epitopes are combined with a linker, with the Fab of the first antibody on one side of the dimeric Fc and the scFv of the second antibody on the other side. It may also be a bispecific molecule linked via The bispecific molecule is in a format in which Fab and scFv, each containing a mutation downstream thereof that forms a heterodimer, are assembled in a heterozygous manner. Such a bispecific molecule is called a hybrid type bispecific molecule or a hybrid type (FIG. 7(7a)). In the present invention, for example, a hybrid type consisting of anti-HLA-A2/NY-ESO Fab and anti-CD3 scFv can be used.
  • it may be a bispecific molecule in which the Fab of the first antibody and the scFv of the second antibody are bound to one side of the Fc of the dimer via a linker.
  • Fab may be bound to Fc and scFv may be bound to the Fab, or scFv may be bound to Fc and Fab may be bound to the scFv.
  • a Fab is attached and an scFv is attached to the Fab.
  • Fab and scFv may be linked to the variable region of Fab via a linker.
  • the bispecific molecule has a format in which an scFv and a Fab are connected by a linker, and an Fc containing a mutation that forms a heterodimer is associated downstream of the scFv and Fab.
  • Such a bispecific molecule is called an scFv-Fab-heterodimer Fc-type bispecific molecule or a scFv-Fab-heterodimer Fc-type molecule (FIG. 7(7c)).
  • an scFv-Fab-heterodimer Fc type consisting of anti-CD3 scFv and anti-HLA-A2/NY-ESO Fab may be used.
  • taFv ( Figure 5 (5c)), which has a format in which two types of scFv, the first antibody and the second antibody, are connected with a linker, is attached to one of the Fc of the dimer via a linker or via a linker. It is also possible to directly combine the two.
  • a bispecific molecule is called a taFv-heterodimer Fc type bispecific molecule or a taFv-heterodimer Fc type molecule (FIG. 5(5d)).
  • the bispecific molecule is in a format in which Fc containing a mutation that forms a heterodimer downstream of taFv is heteroassociated.
  • the order of linkage of the first antibody and second antibody in taFv is not limited, but if the order of linkage of the first antibody and second antibody in taFv is reversed, the first bispecific molecule is converted into a taFv-heterogeneous molecule.
  • the taFv(inversed)-heterodimer Fc type also referred to as the taFv(inversed)-Fc type).
  • Figure 7 (7a) shows the structure of the hybrid type bispecific molecule
  • Figure 7 (7b) shows the structure of the dual type bispecific antibody
  • Figure 7 (7c) shows the structure of the scFv-Fab-heterodimer Fc type bispecific antibody.
  • scFv is shown in Figure 5 (5a)
  • Fab is shown in Figure 5 (5b)
  • taFv is shown in Figure 5 (5c)
  • taFv-heterodimer Fc type bispecific antibody is shown in Figure 5 (5d)
  • Figure 5 (5e) is The structure of the taFv-Fab-heterodimer Fc type bispecific antibody is shown.
  • FIG. 8 (8a) shows a taFv-heterodimer Fc type bispecific antibody (same as Fig. 5 (5d))
  • Fig. 8 (8b) shows a taFv (inversed) - heterodimer Fc type bispecific antibody
  • Figure 9 (9a) shows the first polypeptide contained in the taFv (inversed)-heterodimer Fc type bispecific antibody
  • Figure 9 (9b) shows the first polypeptide contained in the taFv (inversed) - heterodimer Fc type bispecific antibody.
  • the bispecific antibody of the present invention has a structure in which multiple polypeptides are associated.
  • anti-HLA-A2/NY-ESOscFv and anti-CD3scFv taFv may be used as the taFv.
  • the taFv-heterodimer Fc type bispecific antibody preferably comprises (a) an scFv that specifically binds to HLA/NY-ESO, an scFv that specifically binds to CD3, from the N-terminus to the C-terminus; and a first polypeptide comprising in that order an immunoglobulin Fc region (i), and a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii), more preferably (b)
  • the first polypeptide and the second polypeptide are associated in Fc region (i) and Fc region (ii).
  • the Fc regions of the first polypeptide and the second polypeptide may include mutations to form a heterodimer.
  • An example of a taFv-heterodimer Fc type bispecific antibody is shown in FIG. 5(5d).
  • the Fc region (i) portion of the first polypeptide and the Fc region (ii) portion of the second polypeptide shown in black bind, and the first polypeptide and the second polypeptide are meeting.
  • Figure 6 (6a) shows the first polypeptide
  • Figure 6 (6b) shows the second polypeptide.
  • the scFv shown in white is anti-HLA-A2/NY-ESO scFv
  • the scFv shown with diagonal lines in the upper right corner is anti-CD3 scFv.
  • the first polypeptide contained in the more preferred taFv-heterodimer Fc type bispecific antibody of the present invention has the amino acid sequence from the 21st to the 511th amino acid sequence represented by SEQ ID NO: 32, and the amino acid sequence represented by SEQ ID NO: 34.
  • 21st to 511th amino acid sequence 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 42
  • the first polypeptide contained in the even more preferred taFv-heterodimer Fc type bispecific molecule includes SEQ ID NOs: 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 32, 52, and 53 include the 529th to 745th amino acid sequence of the amino acid sequence represented by 54, or the 534th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55 or 56, and furthermore, The first polypeptide contained in the preferred taFv-heterodimer Fc-type bispecific molecule has the amino acid sequence from positions 21 to 745 of the amino acid sequence represented by SEQ ID NO: 32, and the amino acid sequence represented by SEQ ID NO: 34.
  • 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40 amino acid sequence of 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 41, represented by SEQ ID NO: 42 (21st to 745th amino acid sequence of the amino acid sequence, 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 43, 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33) , the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, or the amino acid sequence represented by SEQ ID NO: 54.
  • Consists of amino acid sequence from 20th to 745th Alternatively, it consists of the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55 or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56.
  • the second polypeptide contained in the preferred taFv-heterodimer Fc-type bispecific molecule of the present invention comprises a human antibody-derived hinge region and a mutant Fc, and is even more preferred taFv-heterodimer Fc-type bispecific molecule.
  • the second polypeptide contained in the bispecific molecule comprises the 20th to 246th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31.
  • the first polypeptide consists of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32
  • the second polypeptide consists of the 21st to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 31.
  • NYF-0016 formed by association, a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 84, and a first polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31.
  • NYF-0022 which is formed by association of two polypeptides, a first polypeptide consisting of the amino acid sequence 21st to 745th of the amino acid sequence represented by SEQ ID NO: 35, and a first polypeptide consisting of the amino acid sequence 21st to 246th of the amino acid sequence represented by SEQ ID NO: 31.
  • NYF-0023 which is composed of a second polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 36, a first polypeptide consisting of the amino acid sequence 21st to 745th of the amino acid sequence represented by SEQ ID NO: 36, and a first polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 31.
  • NYF-0027 formed by association of a second polypeptide consisting of positions 21 to 246, a first polypeptide consisting of the amino acid sequence 21 to 745 of the amino acid sequence represented by SEQ ID NO: 37, and a first polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 31 NYF-0035, which is a combination of a second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, and a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38; NYF-0044, which is formed by combining the second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence shown by number 31, and the first consisting of the 21st to 745th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 39.
  • NYF-0045 which is a combination of a polypeptide and a second polypeptide consisting of positions 21 to 246 of the amino acid sequence represented by SEQ ID NO: 31, an amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO: 40;
  • NYF-0047 is a combination of a first polypeptide consisting of a first polypeptide consisting of a polypeptide consisting of a second polypeptide consisting of positions 21 to 246 of the amino acid sequence shown in SEQ ID NO: 31, and a second polypeptide consisting of positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 41.
  • NYF-0048 which is a combination of a first polypeptide consisting of the amino acid sequence of No.
  • 21 and a second polypeptide consisting of the 21st to 246th amino acid sequence of SEQ ID NO: 31, has an amino acid sequence of SEQ ID NO: 42.
  • NYF-0060 which is a combination of a first polypeptide consisting of the 21st to 745th amino acid sequence and a second polypeptide consisting of the 21st to 246th amino acid sequence of SEQ ID NO: 31, represented by SEQ ID NO: 43;
  • NYF-0061 which is formed by an association of a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31 and a second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31, NYF-, which is formed by association of a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33 and a second polypeptide consisting of the 21st to 246th
  • a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52 and a second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31 are associated.
  • NYF-0082 which is formed by association of two polypeptides, can be exemplified as a preferred taFv-heterodimer Fc type bispecific antibody of the present invention. Furthermore, a first polypeptide consisting of the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54 and a second polypeptide consisting of the 20th to 246th amino acid sequence of SEQ ID NO: 31 are associated. NYZ-0038, a first polypeptide consisting of the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, and a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31.
  • NYZ-0082 formed by association of peptides, a first polypeptide consisting of the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, and the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31.
  • NYZ-0083 which is formed by association of a second polypeptide, can be exemplified as a preferred taFv-heterodimer Fc type bispecific antibody of the present invention.
  • NYF-0023, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, and NYZ-0083 have particularly excellent biological activity, physical properties, etc. suitable.
  • the taFv of the first antibody is directly bound to one of the Fc of the dimer, with or without a linker
  • the Fab of the first antibody or the second antibody is bound to the other Fc, They may be linked directly through a linker or without a linker.
  • the bispecific molecule has a format in which Fab is added upstream of the Fc region (ii) (blacked out) side of the taFv-heterodimer Fc type second polypeptide.
  • Such a bispecific molecule is called a taFv-Fab-heterodimer Fc-type bispecific molecule or a taFv-Fab-heterodimer Fc-type molecule (FIG. 5(5e)).
  • taFv contained in the taFv-Fab-heterodimer Fc type bispecific molecule for example, anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv taFv may be used, and as the Fab, for example, HLA/NY-ESO Fab may be used.
  • the taFv-Fab-heterodimer Fc type bispecific antibody preferably comprises (a) from the N-terminus toward the C-terminus, an scFv that specifically binds to human HLA/NY-ESO, and a scFv that specifically binds to CD3.
  • a first polypeptide comprising a binding scFv and an immunoglobulin Fc region (i) in that order;
  • a second polypeptide comprising an immunoglobulin heavy chain comprising an Fc region (ii); and a second polypeptide comprising an immunoglobulin light chain.
  • FIG. 5(5e) An example of a taFv-Fab-heterodimer Fc type bispecific antibody is shown in FIG. 5(5e), the first polypeptide is shown in FIG. 6(6a), the second polypeptide is shown in FIG. 6(6c), The third polypeptide is shown in Figure 6 (6d). As shown in FIG. 5e, the first polypeptide is shown in FIG. 6(6a), the second polypeptide is shown in FIG. 6(6c), The third polypeptide is shown in Figure 6 (6d). As shown in FIG.
  • the second polypeptide consisting of an immunoglobulin heavy chain including the Fc region (i) portion of the first polypeptide and the Fc region (ii) shown in black is connected to the Fc region (i) of the first polypeptide.
  • the region (i) portion is bound to the Fc region (ii) portion of the second polypeptide, and further, an immunoglobulin light chain is bound to the second polypeptide.
  • Such a preferred taFv-Fab-heterodimer Fc-type bispecific molecule is formed by binding Fab to a taFv-heterodimer Fc-type bispecific molecule comprising taFv and immunoglobulin Fc regions. can.
  • the amino acid sequence contained in the first polypeptide contained in the preferred taFv-Fab heterodimer Fc type bispecific molecule includes the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, SEQ ID NO: The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO.
  • 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, and the amino acid sequence represented by SEQ ID NO: 39 The 21st to 511th amino acid sequence of the amino acid sequence, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 41, The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 42, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 43, the 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33 ⁇ 511th amino acid sequence, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53
  • the first polypeptide contained in the more preferable taFv-Fab heterodimer Fc type bispecific molecule is the amino acid sequence from the 21st to the 745th amino acid sequence represented by SEQ ID NO: 32.
  • Amino acid sequence, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequence represented by SEQ ID NO: 36 The 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, SEQ ID NO: The 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO.
  • 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 42 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 43, and the amino acid sequence represented by SEQ ID NO: 33.
  • the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54 the 21st to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or the amino acid sequence represented by SEQ ID NO: 56.
  • the second polypeptide contained in the preferred taFv-Fab-heterodimer Fc type bispecific antibody of the present invention comprises the variable region, CH1 region and hinge region of a human antibody or humanized antibody heavy chain, as well as mutant Fc.
  • the second polypeptide contained in the more preferred taFv-Fab-heterodimer Fc type bispecific antibody comprises the 20th to 242nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 44. Become.
  • the third polypeptide contained in the preferred taFv-Fab-heterodimer Fc type bispecific antibody of the present invention comprises the variable region and constant region of a human antibody or humanized antibody light chain, and is a more preferred taFv
  • the third polypeptide contained in the -Fab-heterodimer Fc type bispecific molecule comprises the 21st to 131st amino acid sequences represented by SEQ ID NO: 45.
  • variable region and CH1 region of the second polypeptide contained in this preferred taFv-Fab-heterodimer Fc type bispecific molecule and the third polypeptide constitute Fab, and the preferred Fab is anti-HLA/NY- Fab of ESO antibody, for example, Fab of NYA-0001.
  • the scFv included in the scFv-Fab-heterodimer Fc type bispecific antibody for example, anti-HLA-A2/NY-ESO scFv or anti-CD3 scFv may be used, and as the Fab, for example, HLA/NY-ESO Fab or anti-CD3 Fab may be used.
  • the scFv-Fab-heterodimer Fc-type bispecific molecule preferably comprises (a) from the N-terminus towards the C-terminus, an scFv that specifically binds to human HLA/NY-ESO, and a scFv that specifically binds to CD3; A first polypeptide comprising, in that order, the variable region and constant region CH1 of an antibody heavy chain to be bound and an immunoglobulin Fc region (i), and a second polypeptide comprising an immunoglobulin hinge region and Fc region (ii).
  • the first polypeptide and the second polypeptide have an Fc region (i) and an Fc region ( ii), the first polypeptide is associated with the third polypeptide (the antibody light chain) in the variable region and constant region CH1 of the antibody heavy chain.
  • the Fc regions of the first polypeptide and the second polypeptide may be wild type or may contain mutations that form a heterodimer.
  • An example of a scFv-Fab-heterodimer Fc type bispecific molecule is shown in Figure 7 (7c). The right half of FIG.
  • the first polypeptide and the third polypeptide is the first polypeptide and the third polypeptide, and the left half is the second polypeptide.
  • the Fc region (i) portion of the first polypeptide and the Fc region (ii) portion of the second polypeptide shown in black associate with each other, and the first polypeptide and the third polypeptide are meeting.
  • the scFv indicated by diagonal lines in the upper right is anti-HLA-A2/NY-ESO scFv
  • the Fab indicated by white, checkered patterns, and horizontal lines is anti-CD3 Fab.
  • the amino acid sequence contained in the first polypeptide contained in the preferred scFv-Fab-heterodimer Fc type bispecific antibody is the 21st to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 57, More preferably, the 20th to 724th amino acid sequences can be exemplified.
  • amino acid sequences contained in the first polypeptide contained in the preferred scFv-Fab-heterodimer Fc type bispecific antibody include the 21st to 389th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 60.
  • the amino acid sequence more preferably the 20th to 719th amino acid sequence, can be exemplified.
  • amino acid sequences contained in the first polypeptide contained in the preferred scFv-Fab-heterodimer Fc type bispecific antibody include the 21st to 389th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 61.
  • the amino acid sequence more preferably the 20th to 719th amino acid sequence, can be exemplified.
  • the second polypeptide contained in the preferred scFv-Fab-heterodimer Fc type bispecific antibody of the present invention comprises a hinge region derived from a human antibody and a mutant Fc, and an even more preferred scFv-Fab-
  • the second polypeptide contained in the heterodimer Fc type bispecific molecule comprises the 20th to 246th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31.
  • the third polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a light chain derived from a human antibody
  • the third polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule comprises Examples of the third polypeptide included in the heavy specificity molecule include the 21st to 127th amino acid sequence, more preferably the 21st to 233rd amino acid sequence of the amino acid sequence shown by SEQ ID NO: 58. be able to.
  • a first polypeptide consisting of the 20th to 724th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 57 a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31, And NYZ-1010, which is formed by association of a third polypeptide consisting of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 58, is used as a preferred scFv-Fab-heterodimer Fc type bispecific of the present invention. It can be exemplified as a molecule.
  • a first polypeptide consisting of the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 60 a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31, and NYZ-1007, which is formed by association of a third polypeptide consisting of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 58, is also suitable as a preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention.
  • I can give an example.
  • a first polypeptide consisting of the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 61 a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31, and NYZ-1017, which is formed by association of a third polypeptide consisting of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 58, is also suitable as a preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention.
  • I can give an example.
  • One, two or more of the peptides comprised in the bispecific molecule of the invention may be "deletions" as described above, i.e. at their carboxyl terminus, especially at the carboxyl end derived from the antibody heavy chain. At the terminus, one or two (or more) amino acids may be mutated (including deletions).
  • the carboxyl terminal of the first polypeptide contained in NYZ-1010 which is one of the preferred scFv-Fab-heterodimer Fc type bispecific molecules of the present invention, is the 724th amino acid sequence of SEQ ID NO: 57. It may be Lys, Gly at position 723 with one amino acid deleted, or a mixture containing them.
  • the carboxyl terminal of the amino acid sequence of the second polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention has one amino acid deletion at Lys at position 246 of SEQ ID NO: 84. or a mixture containing them.
  • the scFv and Fab contained in the bispecific molecule of the present invention are preferably those of a humanized antibody or a human antibody, and the Fc is preferably that of a human antibody.
  • variable regions contained in the bispecific molecule of the present invention the heavy chain variable region and the light chain variable region may be linked in this order from the amino terminal side of the antibody, or the light chain variable region and the heavy chain variable region may be linked in this order from the amino terminal side of the antibody. They may be linked in the order of variable regions.
  • a linker may be present between both variable regions (optionally).
  • the variable region may also have a glycine residue at the amino terminus (optionally).
  • a linker, a FLAG tag, and/or a His tag may (optionally) be attached to the carboxyl terminus of the variable region on the carboxyl terminal side.
  • One preferred embodiment is one in which the heavy chain variable region, the first linker, the light chain variable region, the second linker, the FLAG tag, and the His tag are linked in this order from the amino terminal. .
  • Linkers include single-chain polypeptides or single-chain oligopeptides, or synthetic products such as PEG, nucleotides, sugar chains, and compounds.
  • any known linker can be used without particular limitation as long as it connects two polypeptides.
  • the length of the linker is, for example, 5 to 30 amino acids in the case of a peptide linker. If the bispecific molecule includes multiple linkers, all peptide linkers may be of the same length, or peptide linkers of different lengths may be used.
  • Examples of the peptide linker include repeats of (Gly ⁇ Gly ⁇ Gly ⁇ Gly ⁇ Ser), but one to several amino acid residues different from Gly and Ser may be added to these.
  • preferred ones are taFv-heterodimer Fc type, taFv-Fab-heterodimer Fc type, and The scFv-Fab-heterodimer Fc type is preferred, and the more preferable one is the taFv-heterodimer Fc type, which is located in the order of anti-HLA/NY-ESO scFv and anti-CD3 scFv from the N-terminus to the C-terminus.
  • the inverted type (taFv-heterodimer Fc type) is more suitable than the opposite type (taFv (inverted)-heterodimer Fc type).
  • Another more preferred type is the scFv-Fab-heterodimer Fc type.
  • the present invention includes an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence contained in the molecule of the present invention. Also included are molecules that contain and bind to HLA/NY-ESO and preferably also bind to CD3.
  • the present invention includes amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to the amino acid sequences contained in the molecules of the present invention. and binds to HLA/NY-ESO and preferably also binds to CD3.
  • the antibodies of the present invention, binding fragments thereof, and multispecific antibodies containing them have excellent biological activity, physicochemical properties (hereinafter referred to as "physical properties"), safety, pharmacokinetics, etc.
  • biological activity or its index As biological activity or its index, antigen binding activity, in vitro cytotoxic activity, in vivo antitumor activity, etc. can be exemplified.
  • the dissociation constant (KD value) for HLA/NY-ESO is 100 nM or less or 50 nM or less, preferably 20 nM or less or 10 nM or less, more preferably 5 nM or less.
  • the EC 50 value of cytotoxic activity exerted against endogenous human NY-ESO expressing cell lines U266B1 and/or NCI-H1703 using human peripheral blood mononuclear cells as effector cells is 20 nM.
  • it is preferably 10 nM or less, more preferably 5 nM or less (the method described in Example 8 of WO2021/200857 can be exemplified as an example of measuring and calculating in vitro cytotoxic activity, but is not limited thereto).
  • 0.1 mL of a suspension of 6 ⁇ 10 7 cells/mL of human squamous cell lung cancer cell line NCI-H1703 was subcutaneously transplanted into NOG mice, and 4 days later, 3.75 ⁇ 10 cells/mL of human peripheral blood mononuclear cells were transplanted subcutaneously into NOG mice.
  • 0.2 mL of 7 cells/mL suspension was implanted into the tail vein, and after 14 days, the antibody was administered three times once a week. Tumor growth inhibitory activity was measured relative to the control group administered with vehicle.
  • HMWS aggregates
  • HMWS is one of the major impurities and is an item that should be particularly strictly controlled because it is also involved in immunogenicity risk and reduction in drug efficacy. Management of impurities needs to be evaluated not only during manufacturing, but also including stability (presence or absence of increase) over time during the manufacturing process and after manufacturing.
  • the shelf life of a drug is determined based on the results of long-term stability tests, so antibodies that are stable over time can have a longer shelf life. Therefore, the physicochemical properties in the present invention that serve as indicators for selecting suitable antibodies for biopharmaceuticals include acid resistance (HMWS production inhibition, etc.) and solution stability (HMWS production inhibition, etc.).
  • a host cell suitable for producing the antibody of the present invention, a binding fragment thereof, or a molecule containing them, such as Expi293F cells may be used to introduce a gene encoding the amino acid sequence contained therein. Examples include high yield and high yield in a culture of recombinant cells.
  • Suitable antibodies of the invention, antigen-binding fragments thereof and multispecific antibodies containing them with their physico-chemical property characteristics allow their solution to be exposed to acidic conditions and their manufacture; For example, it becomes possible or easy to carry out processes such as protein A, chromatography such as ion exchange, and virus inactivation; even if they are used as a solution, the production of HMWS can be suppressed to a low level, so it is easy to carry out their production, formulation, It becomes possible or easy to distribute and preserve pharmaceuticals containing them; it becomes possible to efficiently produce them.
  • the content of HMWS calculated by incubating at pH 3.5, room temperature for 1 hour, and then measuring HMWS by size exclusion chromatography is 5% or less, preferably 2% or less, more preferably is 1% or less (the method described in Example 19-1 of WO2021/200857 can be exemplified as an example of measuring and calculating the HMWS content for acid resistance evaluation, but is not limited thereto).
  • Solution stability is calculated by, for example, dissolving the solution in pH 6.0 consisting of 25mM histidine and 5% sorbitol to a concentration of 25mg/ml, storing it at 25°C for 6 days, and then measuring HMWS by size exclusion chromatography.
  • HMWS The content of HMWS is 20% or less, preferably 10% or less (for measuring and calculating the HMWS content for solution stability evaluation, the method described in Example 19-2 of WO2021/200857 is exemplified) (but not limited to).
  • methods for measuring and calculating production efficiency or yield include, but are not limited to, the methods described in Examples 20 and 21 of WO2021/200857.
  • the immunogenicity is low in the ISPRI web-based immunogenicity screening (EpiVax, Inc), and the risk of side effects such as cytokine production caused by anti-antibodies is low.
  • NYF-0023, NYF-0045, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0082, or NYZ-1010 contained in the bispecific antibody of the present invention can be used as a Balb.
  • no problematic findings were observed regarding blood half-life, weight loss, or other significant toxicity findings.
  • NYZ-0082 or NYZ-1010 was administered once to cynomolgus monkeys, no problematic findings were observed regarding blood half-life, and no changes were observed in general condition, body weight, food intake, body temperature measurement, and No treatment-related changes in plasma cytokine levels were observed.
  • bispecific antibodies obtained in the present invention were administered to Balb/c mice or cynomolgus monkeys, no problematic findings regarding blood half-life were observed.
  • the antibodies, binding fragments and molecules thereof of the present invention having such excellent biological activity, physicochemical properties, safety, pharmacokinetics, etc. can be suitably included in pharmaceutical compositions. .
  • Preferred antibodies or antigen-binding fragments thereof of the present invention having the antigen-binding activity described in (a) and the properties described in (b) include NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA- 2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2031, NYA-2047, NYA-2061, NYA-2143 and NYA-3061, more preferably NYA-2047 , NYA-2061, NYA-2143 and NYA-3061, but are not limited thereto.
  • preferred multispecific antibodies of the present invention having the properties described in (a) to (d) include NYF-0016, NYF-0019, NYF-0022, NYF-0023, NYF-0027, NYF-0035, NYF-0044, NYF-0045, NYF-0047, NYF-0048, NYF-0058, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088 and NYZ-1010, more preferably, Examples include NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, NYZ-1007, NYZ-1010, and NYZ-1017, but are not limited thereto.
  • the "site" to which an antibody binds refers to a partial peptide or partial conformation on an antigen that the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site.
  • Examples of the site on HLA/NY-ESO that the anti-HLA/NY-ESO antibody of the present invention binds to or recognizes include multiple amino acids, partial conformation structures, etc. in the HLA/NY-ESO peptide.
  • Antibodies or binding fragments thereof that bind to the same site are also included in the present invention.
  • An "antibody that binds to the same site” as a certain antibody refers to another antibody that binds to the site on the antigen molecule that the antibody recognizes. If the second antibody binds to a partial peptide or partial three-dimensional structure on the antigen molecule to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same site.
  • the second antibody that binds to the same site on HLA/NY-ESO is highly likely to have the same activity; Dual antibodies are also encompassed by the invention.
  • antibodies that compete with the first antibody of the present invention in binding to HLA/NY-ESO are also encompassed by the present invention, as long as they have the antigen-binding activity described in (a).
  • Such antibodies that bind to the site on HLA/NY-ESO recognized by the monoclonal antibody of the present invention antibodies that compete with the monoclonal antibody of the present invention in binding to HLA/NY-ESO, and binding fragments thereof,
  • one or more of the in vitro cytotoxic activity and in vivo antitumor activity described in (a) and the properties described in (b) to (d), more preferably three or more thereof, Optimal has everything.
  • the binding site of an antibody can be determined by methods well known to those skilled in the art, such as immunoassay methods. For example, a series of peptides are created by appropriately trimming the amino acid sequence of the antigen from the C-terminus or the N-terminus, the reactivity of the antibody against them is examined, the rough recognition site is determined, and then even shorter peptides are synthesized.
  • the binding site can be determined by examining the reactivity of antibodies to those peptides.
  • a specific site or region of the amino acid sequence of an antigen or antigen fragment peptide, etc. may be deleted or substituted with another amino acid sequence, or mutations may be introduced into the amino acid sequence, and the reactivity of antibodies to those peptides may be examined. By doing so, the binding site can be determined.
  • Antigen fragment peptides can be prepared using techniques such as genetic recombination and peptide synthesis.
  • the binding site of such an antibody is determined by identifying amino acid residues on the antigen adjacent to the antibody using X-ray structural analysis. be able to.
  • amino acid residues on the antigen that have an interaction distance with the antibody can be identified by binding and crystallizing an antibody or its fragment and an antigen or its fragment, and performing structural analysis.
  • the interaction distance is 8 ⁇ or less, preferably 6 ⁇ or less, and more preferably 4 ⁇ or less.
  • One or more of the amino acid residues that have such an interaction distance with the antibody can constitute the antigen-binding site (epitope) of the antibody. When there are two or more such amino acid residues, each amino acid does not need to be adjacent to each other in the primary sequence.
  • the anti-HLA/NY-ESO antibody or binding fragment thereof of the present invention specifically recognizes multiple amino acids present in the amino acid sequence of HLA/NY-ESO.
  • An antibody or binding fragment thereof that recognizes the plurality of amino acids competes with the antibody or binding fragment thereof of the present invention in binding to HLA/NY-ESO, or has an interaction distance with the plurality of amino acids, Included in the present invention.
  • multispecific antibodies comprising such antibodies or binding fragments thereof are also encompassed by the invention.
  • Multispecific antibodies of the invention can be used in combination with immune checkpoint inhibitors.
  • immune checkpoint inhibitor refers to a drug that inhibits the immunosuppressive system and activates tumor immunity, and has the same meaning as a "compound that inhibits immune checkpoint molecules.”
  • the immune checkpoint inhibitor used in the present invention is not particularly limited, but preferably includes anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-TIGIT antibody. , more preferably anti-PD-1 antibodies and anti-PD-L1 antibodies.
  • anti-PD-1 antibody refers to an antibody that specifically binds to PD-1 (Programmed cell death-1; CD279; PDCD1), preferably an antibody that specifically binds to PD-1 and its binding partner. This refers to an antibody that has the effect of reducing, inhibiting, and/or interfering with certain signal transduction resulting from interaction with PD-L1 or PD-L2.
  • the anti-PD-1 antibody used in the present invention is not particularly limited as long as its clinical efficacy and safety have been confirmed, but Nivolumab (International Publication No. 2006/121168) is preferably used. etc.) and pembrolizumab (WO 2008/156712 etc.).
  • Nivolumab International Publication No. 2006/121168
  • pembrolizumab WO 2008/156712 etc.
  • commercially available research anti-PD-1 antibodies e.g. clone RMP1-14
  • RMP1-14 research anti-PD-1 antibodies
  • anti-PD-L1 antibody refers to an antibody that specifically binds to PD-L1 (Programmed cell death ligand 1; CD274; B7-H1), and preferably PD-L1 and its binding. This refers to an antibody that has the effect of reducing, inhibiting, and/or interfering with signal transduction resulting from interaction with partner PD-1 or B7.1 (CD80).
  • the anti-PD-L1 antibody used in the present invention is not particularly limited as long as clinical efficacy and safety have been confirmed, but preferably atezolizumab (International Publication No.
  • anti-CTLA-4 antibody refers to an antibody that specifically binds to CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4; CD152), preferably CTLA-4 and its binding partner.
  • the anti-CTLA-4 antibody used in the present invention is not particularly limited as long as clinical efficacy and safety have been confirmed, but preferably Ipilimumab (International Publication No. 2001/014424) ), tremelimumab (WO 2000/037504, etc.), spartalizumab (WO 2015/112900, etc.), cemiplimab (WO 2015/196051, etc.) I can give an example.
  • anti-TIGIT antibody refers to an antibody that specifically binds to TIGIT (T cell immunoreceptor with Ig and ITIM domains), and preferably the interaction between TIGIT and its binding partner, CD155. Indicates an antibody that has the effect of reducing, inhibiting, and/or interfering with signal transduction resulting from.
  • the anti-TIGIT antibody used in the present invention is not particularly limited as long as clinical efficacy and safety are confirmed, but preferably Tiragolumab (International Publication No. 2017/053748), Vibostolimab (International Publication No. 2016/028656) can be exemplified.
  • Tiragolumab International Publication No. 2017/053748
  • Vibostolimab International Publication No. 2016/028656
  • commercially available anti-TIGIT antibody for research use e.g. clone 1B4 was used. etc. can also be used.
  • the antigen-binding fragment of the antibody is also included in the scope of the "antibody”.
  • the immune checkpoint inhibitor used in combination with the multispecific antibody may be an antigen-binding fragment of any of the antibodies described above, and the antigen-binding fragment may contain other moieties.
  • chemotherapeutic agent refers to a chemically synthesized drug among compounds having anticancer or antitumor activity.
  • chemotherapeutic agents used in the present invention are not particularly limited, but preferably include topoisomerase inhibitors, microtubule inhibitors, platinum agents, DNA demethylating agents, anticancer antibiotics, and alkylating agents.
  • topoisomerase inhibitors include irinotecan, topotecan, etoposide, and sacituzumab govitecan, which is conjugated with SN-38, an active metabolite of irinotecan, as a drug complex.
  • microtubule inhibitors include paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, albumin-suspended paclitaxel (Nab-paclitaxel), and the like.
  • platinum preparations include oxaliplatin, carboplatin, cisplatin, nedaplatin, and the like.
  • DNA demethylating agents include azacytidine, decitabine, and the like.
  • anticancer antibiotics include doxorubicin, bleomycin, liposomal doxorubicin, and the like.
  • alkylating agents include ifosfamide, cyclophosphamide, dacarbazine, and the like.
  • chemotherapeutic agents used in combination with the multispecific antibodies of the present invention are not limited to these.
  • compositions and therapeutic methods comprising a combination of a multispecific molecule and an immune checkpoint inhibitor or chemotherapeutic agent.
  • the pharmaceutical composition and treatment method of the present invention allows the multispecific molecule and the immune checkpoint inhibitor or chemotherapeutic agent to be contained as active ingredients in separate formulations and administered at the same time or at different times.
  • the multispecific molecule and the immune checkpoint inhibitor or chemotherapeutic agent may be administered as active ingredients in a single formulation. It's okay.
  • the multispecific molecule according to the present invention may be contained as an active ingredient in a single preparation and administered for the treatment of diseases that are improved by the action of activating anti-tumor immunity.
  • the pharmaceutical composition and treatment method of the present invention can be used for the treatment of cancer, preferably lung cancer (including non-small cell lung cancer), urothelial cancer, colorectal cancer (colorectal cancer), etc.
  • gastric cancer sometimes called gastric adenocarcinoma
  • gastroesophageal junction adenocarcinoma gastrointestinal stromal tumor
  • Cervical cancer esophageal cancer
  • squamous cell carcinoma peritoneal cancer
  • liver cancer hepatocellular carcinoma
  • endometrial cancer uterine cancer
  • salivary gland cancer kidney cancer
  • vulvar cancer thyroid cancer
  • penile cancer leukemia, malignant lymphoma, selected from the group consisting of plasmacytoma, myeloma, neuroepithelial tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, cholangiocarcinoma, mesothelioma, Paget's disease, and sar
  • the pharmaceutical composition and treatment method of the present invention can be selected and used as a drug for drug therapy, which is the main treatment method for cancer, and as a result, it slows down the growth of cancer cells and inhibits their proliferation. It can suppress and even destroy cancer cells. Through these actions, relief from cancer-related symptoms and improvement of QOL can be achieved in cancer patients, and therapeutic effects can be achieved while preserving the lives of cancer patients. Even if cancer cells are not destroyed, suppression and control of cancer cell proliferation can help cancer patients achieve a higher quality of life and longer survival.
  • the pharmaceutical composition and treatment method of the present invention can also be used as a drug in combination with other therapies in adjuvant therapy, such as surgery, radiotherapy, hormone therapy, etc.
  • the pharmaceutical composition and treatment method of the present invention can also be expected to have preventive effects such as suppressing the proliferation and even destroying microscopic metastatic cancer cells.
  • it can be expected to have the effect of suppressing and destroying cancer cells present in body fluids during the metastasis process, and the effect of suppressing and destroying minute cancer cells immediately after implantation in any tissue. Therefore, it can be expected to suppress and prevent cancer metastasis, especially after surgical removal of cancer.
  • the pharmaceutical composition and treatment method of the present invention can be applied to patients as a systemic therapy, and can also be applied locally to cancer tissues to expect therapeutic effects.
  • compositions of the invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients.
  • the substance to be used in the pharmaceutical composition of the present invention can be appropriately selected from pharmaceutical additives and others commonly used in this field, depending on the dosage and concentration.
  • the pharmaceutical compositions typically include one or more pharmaceutical carriers (eg, sterile liquids).
  • Liquids here include, for example, water and oils (oils of petroleum, animal, vegetable or synthetic origin). The oil may be, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a more typical carrier when the pharmaceutical composition is administered intravenously.
  • Aqueous saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.
  • Appropriate pharmaceutical excipients can be appropriately selected from those known in the art.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of suitable pharmaceutical carriers include E. W. Martin, "Remington's Pharmaceutical Sciences". The formulation will depend on the mode of administration.
  • routes of introduction can include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.
  • Administration can be, for example, by infusion or bolus injection.
  • administration of the multispecific molecules and immune checkpoint inhibitors or chemotherapeutic agents used in the invention is by injection.
  • Parenteral administration is the preferred route of administration.
  • the pharmaceutical composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the pharmaceutical composition can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • a solubilizing agent such as lignocaine to ease pain at the site of the injection.
  • the above ingredients may be present separately or together in unit dosage form, e.g., as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. Mixed and supplied either.
  • the pharmaceutical composition When the pharmaceutical composition is in a form to be administered by injection, it can be dosed, for example, in an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration.
  • the pharmaceutical composition and treatment method of the present invention may contain a cancer therapeutic agent other than the multispecific molecule and immune checkpoint inhibitor or chemotherapeutic agent according to the present invention.
  • the pharmaceutical composition and treatment method of the present invention can also be administered in combination with other cancer therapeutic agents, thereby enhancing the antitumor effect.
  • Other cancer therapeutic agents used for this purpose may be administered to an individual simultaneously with the pharmaceutical composition of the present invention, separately, or consecutively, or may be administered at different intervals. It's okay.
  • Examples of such cancer therapeutic agents include Pemetrexed, Sorafenib, Everolims, Tanespimycin, and Bevacizumab, but these drugs have antitumor activity. There are no limitations.
  • the molecular target drugs used in the present invention are not particularly limited, but are preferably VEGF inhibitors, FGFR inhibitors, and multikinase inhibitors.
  • Preferred examples of VEGF inhibitors include bevacizumab, ramucirumab, Trap, axitinib, etc. can be exemplified.
  • Suitable examples of FGFR inhibitors include pemigatinib, futibatinib, and the like.
  • Suitable examples of multikinase inhibitors include sorafenib, sunitinib, pazopanib, regorafenib, lenvatinib, and the like.
  • the chemotherapeutic agents used in combination with the multispecific antibodies of the present invention are not limited to these.
  • compositions can be formulated as lyophilized or liquid formulations with the selected composition and required purity.
  • preparation may contain suitable additives used in this field.
  • liquid preparations can be formulated as liquid preparations containing various formulation additives used in this field.
  • the composition and concentration of the pharmaceutical composition will also vary depending on the method of administration, the multispecific molecules and immune checkpoint inhibitors or chemotherapeutic agents contained in the pharmaceutical compositions of the present invention have an affinity for the antigen, i.e. In terms of dissociation constant (Kd value), the higher the affinity (lower the Kd value), the more effective the drug can be exerted even at a small dose. Therefore, the dosage of the multispecific molecule and immune checkpoint inhibitor or chemotherapeutic agent can also be set based on the affinity situation with the antigen.
  • the dose is, for example, 0.0001 mg to 100 mg.
  • a predetermined dose may be administered once every 1 to 180 days, or may be divided into two, three, four or more doses per day and administered at appropriate intervals.
  • the administration method includes, for example, a method in which 0.001 mg/kg to 8 mg/kg is administered once every three weeks.
  • administration may be done once every three weeks (q3w), but it may also be administered once a week (q1w), once every two weeks (q2w), or once every four weeks (q4w). .
  • q3w once every three weeks
  • q1w once every two weeks
  • q4w once every four weeks
  • each operation related to genetic manipulation is performed by the method described in the experimental manual used by those skilled in the art, or if commercially available reagents or kits are used, the instructions for the commercially available product are used. I followed.
  • Example 1 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Paclitaxel on human squamous cell lung cancer cell line
  • Human squamous cell lung cancer cell line NCI-H1703 (ATCC ) was prepared at 6 ⁇ 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • Example 2 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Nab Paclitaxel on human squamous cell lung cancer cell line
  • Human squamous cell lung cancer cell line NCI-H1703 ( ATCC) was prepared at 6 ⁇ 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • Example 3 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-PD-1 antibody Pembrolizumab on human lung adenocarcinoma cell lines Human lung adenocarcinoma Cell line NCI-H522 (ATCC) was prepared at 5 x 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4-6 weeks old). I did it (Day 0). After about 2 weeks, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • n 5 in each group, and human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells to 5 x 10 7 cells/mL with PBS. 0.2 mL was implanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg/kg) was administered into the tail vein. Pembrolizumab (Merck, 10 mg/kg) was also administered into the tail vein. Administration was performed on Day 25, Day 32, Day 39 (NYZ-1010), or Day 28, Day 32, Day 35, Day 39, Day 42 (Pembrolizumab). Tumor Growth Inhibition (%) was calculated using the formula shown below.
  • TGI 53 days after transplantation was 50.5% in the NYZ-1010 monotherapy group and 3.1% in the Pembrolizumab monotherapy group, whereas it was 79.0% in the NYZ-1010 and Pembrolizumab combination group. It was shown that the antitumor effect was enhanced by combined use.
  • Example 4 Generation of human CD3 ⁇ knock-in mice
  • the anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule binds to human CD3 ⁇ but does not cross-react to rodent CD3 ⁇ . Therefore, in order to enable in vivo evaluation of the anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, human CD3 ⁇ knock-in mice were generated.
  • a vector was created in which a human CD3 ⁇ sequence that binds CRISPR-Cas9 and an anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule was knocked into mouse CD3 ⁇ , and the vector was injected into fertilized eggs of C57BL/6J mice using the microinjection method. Introduced.
  • the fertilized eggs after injection were transplanted into the oviduct or uterus of a female mouse, and the tail tissues of the resulting offspring were biopsied and subjected to PCR and sequence analysis. Individuals with the desired mutation were selected and subjected to subsequent experiments.
  • Example 5 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYF-0016) and anti-mouse PD-1 antibody using human CD3 ⁇ knock-in mice Introduced from National Cancer Institute MC-38 NY-SCT cells were used, in which HLA-A2, NY-ESO, and ⁇ 2M Single Chain Trimer (abbreviated as NY-SCT) were introduced into mouse colon cancer cell line MC-38 using a retrovirus vector. This cell expresses HLA-A2/NY-ESO on the cell membrane.
  • NY-SCT Single Chain Trimer
  • MC-38 NY-SCT was prepared at 8 ⁇ 10 6 cells/mL in PBS (Wako) and injected subcutaneously into the human CD3 ⁇ knock-in mouse (female, 6-7 weeks old) prepared in Example 4 at 0. 1 mL was transplanted (Day 0). From Day 7, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • TGI 17 days after transplantation was 31.5% in the NYF-0016 monotherapy group and 35.3% in the anti-mouse PD-1 antibody monotherapy group, whereas NYF-0016 and anti-mouse PD-1 antibody therapy In the combination group, it was 57.5%, indicating that the antitumor effect was enhanced by the combination.
  • Example 6 In vivo combination of Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) with anti-PD-1 antibody Pembrolizumab and multikinase inhibitor lenvatinib against human lung adenocarcinoma cell lines Effect Human lung adenocarcinoma cell line NCI-H522 (ATCC) was prepared at 5 x 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and cultured in NSG mice (female, 4-6 weeks old). 0.1 mL was subcutaneously transplanted (Day 0). After about 3 weeks, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • CD3/CD28 Dynabeads ThermoFisher
  • Tumor Growth Inhibition (%) was calculated using the formula shown below.
  • Example 7 Preparation of BALB/C strain human CD3 ⁇ knock-in mouse
  • the C57BL/6J mouse strain human CD3 ⁇ knock-in mouse produced in Example 4 was backcrossed with the BALB/C strain to generate a BALB/C strain human CD3 ⁇ knock-in mouse.
  • PCR and sequence analysis were performed on the tail tissues of the resulting offspring. Individuals with a high homosubstitution rate were selected and bred, and used for subsequent experiments.
  • Example 8 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse PD-1 antibody using BALB/C strain human CD3 ⁇ knock-in mice From ATCC The introduced mouse colon cancer cell line CT26.
  • CT26 NY-SCT was prepared with PBS (Wako) at 1 x 10 7 cells/mL, and injected subcutaneously into the BALB/C strain human CD3 ⁇ knock-in mouse (female, 7 to 9 weeks old) prepared in Example 7. 0.1 mL was transplanted (Day 0). From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • n 5 to 10 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg/kg) was injected into the tail vein. Administered intravenously. In addition, anti-mouse PD-1 antibody (clone RMP1-14, BioX Cell, 5 mg/kg) was administered intraperitoneally. Administration was carried out on Day 8. Tumor Growth Inhibition (%) was calculated using the formula shown below.
  • FIG. 73 (1) The results of administration of NYZ-1010 alone, anti-mouse PD-1 antibody alone, and combined administration of NYZ-1010 and anti-mouse PD-1 antibody are shown in FIG. 73 (1).
  • TGI 21 days after transplantation was 70.8% in the NYZ-1010 alone administration group and 38.0% in the anti-mouse PD-1 antibody alone administration group, whereas NYZ-1010 and anti-mouse PD-1 antibody In the combination group, it was 84.8%, indicating that the antitumor effect was enhanced by the combination.
  • FIG. 73(2) Tumors remained in each single administration group, and the drug efficacy was limited (gray solid line and thin broken line, respectively), whereas presumed tumors remained in 3 out of 10 patients in the NYZ-1010 and anti-mouse PD-1 antibody combination group. The volume showed 0 mm3 , and complete regression was maintained thereafter (black circle, solid line).
  • CT26 NY-SCT was retransplanted into completely regressed mice or into BALB/C strain human CD3 ⁇ knock-in mice (hereinafter referred to as Naive mice, female, 7 to 9 weeks old) that had no experience of tumor transplantation.
  • CT26 NY-SCT was prepared at 1 ⁇ 10 7 cells/mL with PBS (Wako), and 0.1 mL was implanted subcutaneously (Day 59). In naive mice, the tumor engrafted and grew (rough dashed line), but in mice with complete regression, the tumor did not engraft, and complete regression was maintained by NYZ-1010 and anti-mouse PD-1 antibody (black circles, solid line), and the tumor It was shown that immune memory for the cells was established.
  • human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells, which were prepared in PBS to a concentration of 5 x 10 7 cells/mL, and 0.2 mL was transplanted into the tail vein.
  • CD3/CD28 Dynabeads ThermoFisher
  • T cells were prepared in PBS to a concentration of 5 x 10 7 cells/mL, and 0.2 mL was transplanted into the tail vein.
  • anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule NYZ-1010, 0.02 mg/kg
  • Durvalumab Advalumab (AstraZeneca, 10 mg/kg) was administered intraperitoneally. Administration was performed on Day 25, Day 32 (NYZ-1010), or Day 28, Day 32, Day 35, Day 39 (Durvalumab). Tumor Growth Inhibition (%) and
  • Example 10 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse PD-L1 antibody using BALB/C strain human CD3 ⁇ knock-in mice
  • Example 8 The CT26 NY-SCT prepared in Example 7 was adjusted to 1 ⁇ 10 7 cells/mL with PBS (Wako), and used in the BALB/C strain human CD3 ⁇ knock-in mouse (female, 6-weeks old) prepared in Example 7. 0.1 mL was subcutaneously transplanted (Day 0). From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • n 5 to 10 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg/kg) was injected into the tail vein. Administered intravenously. In addition, anti-mouse PD-L1 antibody (clone 10F.9G2, BioX Cell, 5 mg/kg) was administered intraperitoneally. Administration was carried out on Day 8. Tumor Growth Inhibition (%) was calculated using the formula shown below.
  • FIG. 75 (1) The results of administration of NYZ-1010 alone, anti-mouse PD-L1 antibody alone, and combined administration of NYZ-1010 and anti-mouse PD-L1 antibody are shown in FIG. 75 (1).
  • TGI 21 days after transplantation was 68.7% in the NYZ-1010 alone administration group and 36.4% in the anti-mouse PD-L1 antibody alone administration group, whereas NYZ-1010 and anti-mouse PD-L1 antibody In the combination group, it was 77.4%, indicating that the antitumor effect was enhanced by the combination.
  • FIG 75 (2) Tumors remained in each monoadministration group, and the drug efficacy was limited (solid gray line and fine dashed line, respectively), whereas estimated tumors in 2 out of 10 patients in the NYZ-1010 and anti-mouse PD-L1 antibody combination group remained. The volume showed 0 mm3 , and complete regression was maintained thereafter (black circle, solid line).
  • CT26 Mock was prepared with PBS (Wako) at 1 ⁇ 10 7 cells/mL, and 0.1 mL was implanted subcutaneously (Day 64). In naive mice, the tumor engrafted and grew (rough dashed line), but in mice with complete regression, the tumor did not engraft, and complete regression was maintained by NYZ-1010 and anti-mouse PD-L1 antibody (black circle, solid line), and the tumor It was shown that immune memory for the cells was established.
  • Example 11 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse CTLA-4 antibody using BALB/C strain human CD3 ⁇ knock-in mice
  • Example 8 The CT26 NY-SCT prepared in Example 7 was prepared at 1 ⁇ 10 7 cells/mL with PBS (Wako), and the BALB/C strain human CD3 ⁇ knock-in mouse (female, 7 to 9 weeks old) prepared in Example 7 was prepared. 0.1 mL was transplanted subcutaneously (Day 0). From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • n 5 to 10 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg/kg) was injected into the tail vein. Administered intravenously. In addition, anti-mouse CTLA-4 antibody (clone 9D9, BioX Cell, 5 mg/kg) was administered intraperitoneally. Administration was carried out on Day 8. Tumor Growth Inhibition (%) was calculated using the formula shown below.
  • Tumor Growth Inhibition (%) 100 - (Estimated Tumor Volume of each group/Estimated Tumor Volume of Vehicle Control x 100)
  • TGI 21 days after transplantation was 70.8% in the NYZ-1010 alone administration group and 12.1% in the anti-mouse CTLA-4 antibody alone administration group, whereas NYZ-1010 and anti-mouse CTLA-4 antibody In the combination group, it was 80.4%, indicating that the antitumor effect was enhanced by the combination.
  • FIG 76(2) Tumors remained in each single administration group, and the drug efficacy was limited (gray solid line and thin broken line, respectively), whereas estimated tumors in 1 out of 10 patients in the NYZ-1010 and anti-mouse CTLA-4 antibody combination group remained. The volume showed 0 mm3 , and complete regression was maintained thereafter (black circle, solid line).
  • CT26 NY-SCT was reimplanted into completely regressed mice and into naive mice (female, 7-9 weeks old).
  • CT26 NY-SCT was prepared at 1 ⁇ 10 7 cells/mL with PBS (Wako), and 0.1 mL was implanted subcutaneously (Day 59).
  • mice In naive mice, the tumor engrafted and grew (rough dashed line), but in mice with complete regression, the tumor did not engraft, and complete regression was maintained by NYZ-1010 and anti-mouse CTLA-4 antibody (black circles, solid line), and the tumor It was shown that immune memory for the cells was established.
  • Example 12 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse TIGIT antibody using human CD3 ⁇ knock-in mouse CT26 NY- produced in Example 8 SCT was prepared at 1 ⁇ 10 7 cells/mL with PBS (Wako), and 0.1 mL was subcutaneously administered to the BALB/C strain human CD3 ⁇ knock-in mouse (female, 7 to 9 weeks old) prepared in Example 7. It was transplanted (Day 0). From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • n 5 to 10 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg/kg) was injected into the tail vein. Administered intravenously. In addition, an anti-mouse TIGIT antibody (clone 1B4, Absolute Antibody, 5 mg/kg) was administered intraperitoneally. Administration was carried out on Day 8. Tumor Growth Inhibition (%) was calculated using the formula shown below.
  • Example 13 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Carboplatin on human squamous cell lung cancer cell line NCI-H1703 (ATCC) ) was prepared at 6 ⁇ 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • TGI 34 days after transplantation was 47.2% in the NYZ-1010 monotherapy group and 15.0% in the Carboplatin monotherapy group, whereas it was 61.6% in the NYZ-1010 and Carboplatin combination group. It was shown that the antitumor effect was enhanced by combined use.
  • Example 14 Effect of in vivo combined use of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and sacituzumab govitecan-hziy on human esophageal cancer cell line
  • Human esophageal cancer cell line OE- 19 (DSMZ) was prepared in PBS containing 50% Matrigel (CORNING) to 5 x 10 7 cells/mL, and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). ). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • human PBMC On Day 7, human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells, which were prepared in PBS to a concentration of 5 x 10 7 cells/mL and 0.2 mL transplanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg/kg) was administered into the tail vein. Administration was performed on Day 7 (NYZ-1010), Day 6, or Day 13 (sacituzumab govitecan-hziy). Tumor Growth Inhibition (%) and Change from baseline (%) were calculated using the formulas shown below.
  • FIG. 79 (1) The results of the single administration of NYZ-1010, the single administration of sacituzumab govitecan-hziy, and the combined administration of NYZ-1010 and sacituzumab govitecan-hziy are shown in FIG. 79 (1).
  • TGI 21 days after transplantation was 79.9% in the NYZ-1010 monotherapy group and 35.4% in the sacituzumab govitecan-hziy monotherapy group, whereas in the NYZ-1010 and sacituzumab govitecan-hziy combination therapy group 96 in group .7%, indicating that the antitumor effect was enhanced by combined use.
  • Example 15 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Doxorubicin on human lung adenocarcinoma cell line
  • Human synovial sarcoma cell line SW982 (ATCC) was prepared in PBS containing 50% Matrigel (CORNING) to a concentration of 5 ⁇ 10 7 cells/mL, and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • human PBMC On Day 41, human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) and proliferated T cells were prepared in PBS to 5x10 7 cells/mL and 0.2 mL was transplanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg/kg) was administered into the tail vein. Administration was performed on Day 41 (NYZ-1010), Day 40, and Day 47 (Doxorubicin). Tumor Growth Inhibition (%) and Change from baseline (%) were calculated using the formulas shown below.
  • Tumor Growth Inhibition (%) 100 - (Estimated Tumor Volume of each group/Estimated Tumor Volume of Vehicle Control x 100)
  • TGI 55 days after transplantation was 71.8% in the NYZ-1010 alone administration group and 25.6% in the Doxorubicin alone administration group, whereas it was 90.2% in the NYZ-1010 and Doxorubicin combination group. It was shown that the antitumor effect was enhanced by combined use.
  • Example 16 In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Decitabine on human squamous cell lung cancer cell line
  • Human squamous cell lung cancer cell line NCI-H1703 (ATCC ) was prepared at 6 ⁇ 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
  • human PBMC On Day 14, human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells, which were prepared in PBS to a concentration of 5 x 10 7 cells/mL, and 0.2 mL was transplanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg/kg) was administered into the tail vein. Administration was performed on Day 14, Day 21 (NYZ-1010), or Day 7, Day 9, Day 11 (Decitabine). Tumor Growth Inhibition (%) and Change from baseline (%) were calculated using the formulas shown below.
  • FIG. 81 (1) The results of NYZ-1010 administration alone, Decitabine administration alone, and NYZ-1010 and Decitabine combination administration are shown in FIG. 81 (1).
  • TGI 32 days after transplantation was 68.0% in the NYZ-1010 alone administration group and 46.7% in the Decitabine alone administration group, whereas it was 93.0% in the NYZ-1010 and Decitabine combination group, It was shown that the antitumor effect was enhanced by combined use.
  • the rate of change from baseline (%) in the tumor volume on Day 32 for each individual with the tumor volume on Day 7 as the baseline was calculated using the formula shown below, and is shown in FIG. 81 (2).
  • the average value of Change from baseline (%) for each group is listed in Table 7.
  • the combination of the antibody provided by the present invention and other drugs enables the treatment or prevention of various cancers.
  • SEQ ID NO: 1 Amino acid sequence of CDRH1 contained in NYA-0001
  • SEQ ID NO: 2 Amino acid sequence of CDRH2 contained in NYA-0001
  • SEQ ID NO: 3 Missing number (SEQ ID NO: of patent application 2022-041253 filed on March 16, 2022) 3 was the amino acid sequence of CDRH3 contained in NYA-0001)
  • SEQ ID NO: 4 Amino acid sequence of CDRL1 contained in NYA-0001 SEQ ID NO: 5: Amino acid sequence of CDRL2 contained in NYA-0001
  • 6 Amino acid sequence of CDRL3 contained in NYA-0001

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Abstract

Provided is a new combination of medicines for treating cancer. A pharmaceutical composition for treating and/or preventing cancer comprises a multi-specific antibody described in [i] below and is used in combination with a compound described in [ii] below: [i] a multi-specific antibody or an antigen-binding fragment thereof; and [ii] an immune checkpoint inhibitor or a chemotherapeutic agent.

Description

多重特異的分子と免疫チェックポイント阻害剤の組み合わせCombination of multispecific molecules and immune checkpoint inhibitors
　本発明は、免疫チェックポイント阻害剤等と組み合わせて使用される、多重特異的分子を含む医薬組成物、治療又は予防方法等に関する。 The present invention relates to pharmaceutical compositions containing multispecific molecules, treatment or prevention methods, etc., which are used in combination with immune checkpoint inhibitors and the like.
　癌の治療では、手術による癌部分の除去、抗癌剤や放射線により癌細胞を殺すことで治癒や延命効果が期待される。しかしながら、多くの癌に対しては単独の抗癌剤治療だけでは十分な効果が得られないため、様々な薬剤との併用により治療が試みられている。抗癌剤にはドキソルビシン、カルボプラチン、タキソール、あるいはカンプトテシンなどの化学療法剤や、イマチニブ、クリゾチニブ、ダサチニブ、ラパチニブなどの分子標的薬、トラスツズマブ、ベバシズマブ、セツキシマブ、ラムシルマブなどの癌治療抗体などがあり、癌の種類や進行度、治療状況により、使われる抗癌剤やその併用療法も決定される。 In the treatment of cancer, curing and survival effects are expected by removing the cancerous part through surgery and killing cancer cells with anticancer drugs and radiation. However, for many cancers, treatment with a single anticancer drug does not provide sufficient effects, and therefore treatments are being attempted using combinations of various drugs. Anticancer drugs include chemotherapy agents such as doxorubicin, carboplatin, taxol, or camptothecin, molecular targeted drugs such as imatinib, crizotinib, dasatinib, and lapatinib, and cancer treatment antibodies such as trastuzumab, bevacizumab, cetuximab, and ramucirumab, depending on the type of cancer. The anticancer drugs and combination therapy to be used are determined depending on the cancer, stage of progression, and treatment status.
　近年標準治療薬になりつつある免疫チェックポイント阻害剤は、免疫抑制系を阻害し、抗腫瘍免疫を活性化する薬剤である（非特許文献１～３）。免疫チェックポイント阻害剤としては、抗ＰＤ－１抗体である、ニボルマブ（Ｎｉｖｏｌｕｍａｂ）（特許文献１）、ペムブロリズマブ（Ｐｅｍｂｒｏｌｉｚｕｍａｂ）（特許文献２）、抗ＰＤ－Ｌ１抗体である、アテゾリズマブ（Ａｔｅｚｏｌｉｚｕｍａｂ）（特許文献３）、デュルバルマブ（Ｄｕｒｖａｌｕｍａｂ）（特許文献４）、アベルマブ（Ａｖｅｌｕｍａｂ）（特許文献５）、抗ＣＴＬＡ－４抗体である、イピリムマブ（Ｉｐｉｌｉｍｕｍａｂ）（特許文献６）、トレメリムマブ（Ｔｒｅｍｅｌｉｍｕｍａｂ）（特許文献７）、スパルタリズマブ　（Ｓｐａｒｔａｌｉｚｕｍａｂ）（特許文献８）、セミプリマブ（Ｃｅｍｉｐｌｉｍａｂ）（特許文献９）、抗ＴＩＧＩＴ抗体である、チラゴルマブ（Ｔｉｒａｇｏｌｕｍａｂ）（特許文献１０）、ビボストリマブ（Ｖｉｂｏｓｔｏｌｉｍａｂ）（特許文献１１）等が知られている。また化学療法剤との組み合わせにより延命効果が認められている事例もある（非特許文献４）。さらにはがん抗原とＣＤ３を標的とした二重特異的抗体との併用効果を検討した事例も報告されている（非特許文献５及び６）。 Immune checkpoint inhibitors, which have become standard therapeutic drugs in recent years, are drugs that inhibit the immunosuppressive system and activate antitumor immunity (Non-patent Documents 1 to 3). Immune checkpoint inhibitors include anti-PD-1 antibodies Nivolumab (Patent Document 1), Pembrolizumab (Patent Document 2), and anti-PD-L1 antibodies Atezolizumab (Patent Document 2). Patent Document 3), Durvalumab (Patent Document 4), Avelumab (Patent Document 5), anti-CTLA-4 antibody Ipilimumab (Patent Document 6), Tremelimumab (Patent Document 7) ), Spartalizumab (Patent Document 8), Cemiplimab (Patent Document 9), anti-TIGIT antibodies Tiragolumab (Patent Document 10), Vibostolimab (Patent Document 11), etc. It has been known. There are also cases in which a combination with chemotherapeutic agents has been shown to have a life-prolonging effect (Non-Patent Document 4). Furthermore, cases have been reported in which the combined effects of a cancer antigen and a bispecific antibody targeting CD3 were investigated (Non-patent Documents 5 and 6).
　しかしながら、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ抗体、及び抗ＣＤ３抗体で構成される二重特異的抗体（特許文献１２）と併用することにより、抗癌作用が増強される薬剤についてはほとんど知られていない。 However, little is known about drugs whose anticancer effects are enhanced when used in combination with a bispecific antibody composed of an anti-HLA-A2/NY-ESO antibody and an anti-CD3 antibody (Patent Document 12). do not have.
特許文献１：　国際公開第２００６／１２１１６８号
特許文献２：　国際公開第２００８／１５６７１２号
特許文献３：　国際公開第２０１０／０７７６３４号
特許文献４：　国際公開第２０１１／０６６３８９号
特許文献５：　国際公開第２０１３／０７９１７４号
特許文献６：　国際公開第２００１／０１４４２４号
特許文献７：　国際公開第２０００／０３７５０４号
特許文献８：　国際公開第２０１５／１１２９００号
特許文献９：　国際公開第２０１５／１９６０５１号
特許文献１０：国際公開第２０１７／０５３７４８号
特許文献１１：国際公開第２０１６／０２８６５６号
特許文献１２：国際公開第２０２１／２００８５７号 Patent Document 1: International Publication No. 2006/121168 Patent Document 2: International Publication No. 2008/156712 Patent Document 3: International Publication No. 2010/077634 Patent Document 4: International Publication No. 2011/066389 Patent Document 5: International Publication No. 2013/079174 Patent Document 6: International Publication No. 2001/014424 Patent Document 7: International Publication No. 2000/037504 Patent Document 8: International Publication No. 2015/112900 Patent Document 9: International Publication No. 2015/196051 Patent Document 10: International Publication No. 2017/053748 Patent Document 11: International Publication No. 2016/028656 Patent Document 12: International Publication No. 2021/200857
非特許文献１：　Ｍｅｎｏｎ　Ｓ．，　ｅｔ　ａｌ．，　Ｃａｎｃｅｒｓ　（２０１６）　８，　１０６．
非特許文献２：　Ｐａｒｄｏｌｌ　ＤＭ．，　Ｎａｔ　Ｒｅｖ　Ｃａｎｃｅｒ　（２０１２）　１２，　２５２－２６４．
非特許文献３：　Ｗｏｌｃｈｏｋ　ＪＤ．，　Ｃｅｌｌ　（２０１５）　１６２，　９３７．
非特許文献４：　Ｍｏｔｚｅｒ，　Ｒ．，　ｅｔ　ａｌ．，　Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｊｏｕｒｎａｌ　ｏｆ　Ｍｅｄｉｃｉｎｅ．　（２０２１）　３８４：１２８９－３００．
非特許文献５：　Ｄｅｅｇｅｎ　Ｐ．，　ｅｔ　ａｌ．，　Ｃｌｉｎｃａｌ　Ｃａｎｃｅｒ　Ｒｅｓｅａｒｃｈ．　（２０２１）　２７：２９２８－３７．
非特許文献６：　Ｔｅｂｅｒｎｅｒｏ　Ｊ．，　ｅｔ　ａｌ．，　Ｊｏｕｒｎａｌ　ｏｆ　Ｃｌｉｎｉｃａｌ　Ｏｎｃｏｌｏｇｙ．　（２０１７）　３５：１５＿ｓｕｐｐｌ，　３００２－３００２． Non-patent document 1: Menon S. , et al. , Cancers (2016) 8, 106.
Non-Patent Document 2: Pardoll DM. , Nat Rev Cancer (2012) 12, 252-264.
Non-Patent Document 3: Wolchok JD. , Cell (2015) 162, 937.
Non-patent document 4: Motzer, R. , et al. , New England Journal of Medicine. (2021) 384:1289-300.
Non-patent document 5: Deegen P. , et al. , Clinical Cancer Research. (2021) 27:2928-37.
Non-patent document 6: Tevernero J. , et al. , Journal of Clinical Oncology. (2017) 35:15_suppl, 3002-3002.
　本発明の一つの課題は抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ抗体、及び抗ＣＤ３抗体で構成される多重特異的抗体および他剤の組合せ、ならびに、他剤と組み合わせて使用される、該抗体又は該抗体を含有する医薬組成物等を提供することである。 One of the objects of the present invention is the combination of a multispecific antibody consisting of an anti-HLA-A2/NY-ESO antibody and an anti-CD3 antibody and other agents, and the use of the antibody or the anti-CD3 antibody in combination with other agents. An object of the present invention is to provide pharmaceutical compositions containing antibodies.
　また、本発明の他の一つの課題は、癌の治療又は予防に使用される抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ抗体、及び抗ＣＤ３抗体で構成される多重特異的抗体および他剤を組み合わせて癌の治療又は予防に使用される、該抗体又は該抗体を含有する医薬組成物を提供することにある。 Another object of the present invention is to combine a multispecific antibody composed of an anti-HLA-A2/NY-ESO antibody and an anti-CD3 antibody used for the treatment or prevention of cancer with other agents to treat or prevent cancer. An object of the present invention is to provide the antibody or a pharmaceutical composition containing the antibody, which is used for the treatment or prevention of.
　さらに、本発明の他の一つの課題は、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ抗体、及び抗ＣＤ３抗体で構成される多重特異的抗体と他剤とを組み合わせて投与することにより、または、該組成物を投与することにより、癌を治療又は予防する方法を提供することにある。  Furthermore, another object of the present invention is to administer a multispecific antibody composed of an anti-HLA-A2/NY-ESO antibody and an anti-CD3 antibody in combination with another agent, or to An object of the present invention is to provide a method for treating or preventing cancer by administering a substance.​
　発明者らは上記課題を解決するために検討を行い、抗癌活性を有する抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ抗体、及び抗ＣＤ３抗体で構成される二重特異的抗体に様々な抗癌剤（例えば、カルボプラチン、パクリタキセル、Ｎａｂパクリタキセルなどの化学療法剤や、ペンブロリズマブなどの免疫チェックポイント阻害剤など）を組み合わせて使用することにより、優れた抗腫瘍効果が得られることを見出し、本発明を完成させた。 The inventors conducted studies to solve the above problems, and added various anticancer drugs (e.g., The present inventors have discovered that excellent antitumor effects can be obtained by using a combination of chemotherapeutic agents such as carboplatin, paclitaxel, and Nab-paclitaxel, and immune checkpoint inhibitors such as pembrolizumab, and have completed the present invention.
　本発明は、
（１）
　下記［ｉｉ］記載の化合物を組み合わせて使用される、下記［ｉ］記載の多重特異性抗体を含むがんの治療及び／又は予防のための医薬組成物：
［ｉ］
　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、若しくは配列番号３で表されるアミノ酸配列において、６番目のアミノ酸がＮであるアミノ酸配列からなる軽鎖ＣＤＲＨ３、
配列番号４で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、若しくは配列番号４で表されるアミノ酸配列において、７番目のアミノ酸がＷ及び／若しくは８番目のアミノ酸がＫであるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、並びに
配列番号６で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３、若しくは配列番号６で表されるアミノ酸配列において、２番目のアミノ酸がＡ若しくはＳであるアミノ酸配列からなる軽鎖ＣＤＲＬ３
を含む、ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片、
並びに、
　配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２若しくは配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＮ又はＳであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２若しくはＡｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＧ、Ｑ、Ｎ、Ｓ又はＡであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、並びに
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
を含む、ＣＤ３に結合する抗体又はその抗原結合断片、
を含む、多重特異性抗体；
［ｉｉ］
　免疫チェックポイント分子を阻害する化合物、又は、化学療法剤、
（２）
　多重特異性抗体が二重特異性抗体である、（１）記載の医薬組成物。 The present invention
(1)
A pharmaceutical composition for the treatment and/or prevention of cancer, comprising the multispecific antibody described in [i] below, which is used in combination with the compound described in [ii] below:
[i]
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
A heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, or a light chain CDRH3 consisting of an amino acid sequence in which the 6th amino acid is N in the amino acid sequence represented by SEQ ID NO: 3,
A light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, or a light chain consisting of an amino acid sequence in which the 7th amino acid is W and/or the 8th amino acid is K in the amino acid sequence represented by SEQ ID NO: 4. CDRL1,
Light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of Figure 11: SEQ ID NO: 5 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, or A light chain CDRL3 consisting of an amino acid sequence represented by SEQ ID NO: 6 in which the second amino acid is A or S.
an antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO,
and,
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8 or heavy chain CDRH2 consisting of the amino acid sequence in which the third amino acid is N or S in the amino acid sequence represented by SEQ ID NO: 8,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top in Figure 12: SEQ ID NO: 11 in Figure 82) or Arg Asp Asp (fifth sequence from the top in Figure 12: SEQ ID NO: 11 in Figure 82) A light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 11) in which the second amino acid is G, Q, N, S or A, and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 12. CDRL3
an antibody that binds to CD3 or an antigen-binding fragment thereof,
a multispecific antibody, including;
[ii]
Compounds that inhibit immune checkpoint molecules or chemotherapeutic agents,
(2)
The pharmaceutical composition according to (1), wherein the multispecific antibody is a bispecific antibody.
（３）
　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片が、下記（ｉ）～（ｖ）のセットからなる群より選択される１つ又は２つ以上のセットのＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含む、（１）又は（２）記載の医薬組成物：
（ｉ）
　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号４で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号６で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
（ｉｉ）
　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号４で表されるアミノ酸配列において、７番目のアミノ酸がＷであるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号６で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
（ｉｉｉ）
　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号３で表されるアミノ酸配列において、６番目のアミノ酸がＮであるアミノ酸配からなる重鎖ＣＤＲＨ３、
配列番号４で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号６で表されるアミノ酸配列において、２番目のアミノ酸がＡであるアミノ酸配列からなる軽鎖ＣＤＲＬ３、
（ｉｖ）
　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号４で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号６で表されるアミノ酸配列において、２番目のアミノ酸がＳであるアミノ酸配列からなる軽鎖ＣＤＲＬ３、並びに
　（ｖ）
　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号４で表されるアミノ酸配列において、７番目のアミノ酸がＷであり、８番目のアミノ酸がＫであるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号６で表されるアミノ酸配列において、２番目のアミノ酸がＡであるアミノ酸配列からなる軽鎖ＣＤＲＬ３、
（４）
　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片が、配列番号１８で表されるアミノ酸配列のアミノ酸番号２１～１４０のアミノ酸配列、又は配列番号６９若しくは配列番号７０で表されるアミノ酸配列と９５％以上の配列同一性を有するアミノ酸配列からなる重鎖可変領域、及び、配列番号１８若しくは配列番号２８で表されるアミノ酸配列のアミノ酸番号１５６～２６６のアミノ酸配列、又は配列番号２３で表されるアミノ酸配列と９５％以上の配列同一性を有するアミノ酸配列からなる軽鎖可変領域を含む、（１）～（３）のいずれか一つに記載の医薬組成物、 (3)
The antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO binds one or more sets of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following sets (i) to (v). The pharmaceutical composition according to (1) or (2), comprising:
(i)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4,
A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6
(ii)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, where the seventh amino acid is W;
A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6
(iii)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
Heavy chain CDRH3 consisting of an amino acid sequence in which the 6th amino acid is N in the amino acid sequence represented by SEQ ID NO: 3;
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4,
A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, where the second amino acid is A;
(iv)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4,
A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
A light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, in which the second amino acid is S, and (v)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3,
Light chain CDRL1 consisting of an amino acid sequence in which the seventh amino acid is W and the eighth amino acid is K in the amino acid sequence represented by SEQ ID NO: 4,
A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, where the second amino acid is A;
(4)
The antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO has the amino acid sequence of amino acids 21 to 140 of the amino acid sequence represented by SEQ ID NO: 18, or the amino acid sequence represented by SEQ ID NO: 69 or SEQ ID NO: 70. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity, and an amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 18 or SEQ ID NO: 28, or the amino acid sequence represented by SEQ ID NO: 23. The pharmaceutical composition according to any one of (1) to (3), comprising a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence of
（５）
　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片が、
　配列番号１３で表されるアミノ酸配列からなる重鎖可変領域
配列番号１５で表されるアミノ酸配列からなる重鎖可変領域、
配列番号２０で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号１７で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号１８で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号１９で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号２２で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号２４で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号２５で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号２６で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号２７で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号２８で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号２９で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号２１で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
配列番号５５で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、若しくは、
　配列番号７１で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、並びに
配列番号１４で表されるアミノ酸配列からなる軽鎖可変領域、
配列番号１６で表されるアミノ酸配列からなる軽鎖可変領域、
配列番号２０で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号１７で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号１８で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号１９で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２２で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２４で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２５で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２６で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２７で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２８で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２９で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２１で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５５で表されるアミノ酸配列の１６１番目～２７１番目のアミノ酸配列からなる軽鎖可変領域、若しくは、
　配列番号７１で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域を含む、（１）乃至（４）のいずれか一つに記載の医薬組成物、
（６）
　配列番号１３で表されるアミノ酸配列からなる重鎖可変領域及び配列番号１４で表されるアミノ酸配列からなる軽鎖可変領域、
配列番号１５で表されるアミノ酸配列からなる重鎖可変領域及び配列番号１６で表されるアミノ酸配列からなる軽鎖可変領域、
配列番号２０で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２０で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号１７で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号１７で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号１８で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号１８で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号１９で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号１９で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２２で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２２で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２４で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２４で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２５で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２５で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２６で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２６で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２７で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２７で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２８で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２８で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２９で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２９で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
配列番号２１で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２１で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、配列番号５５で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号５５で表されるアミノ酸配列の１６１番目～２７１番目のアミノ酸配列からなる軽鎖可変領域、又は、
配列番号７１で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号７１で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域を含む、（１）～（５）記載のいずれか一つに記載の医薬組成物、 (5)
An antibody that binds to HLA/NY-ESO or an antigen-binding fragment thereof,
A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13, a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 14,
A light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29,
A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21,
A light chain variable region consisting of the 161st to 271st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or
The pharmaceutical composition according to any one of (1) to (4), comprising a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71;
(6)
A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 14,
A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29,
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, and a light chain variable region consisting of the 161st to 271st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, Or
A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71. The pharmaceutical composition according to any one of (1) to (5), comprising:
（７）
　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片がｓｃＦｖである、請求項１～６のいずれか一つに記載の医薬組成物。
（８）
　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片が、
　配列番号３０で表されるアミノ酸配列の２１～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号１５で表されるアミノ酸配列からなる重鎖可変領域及び配列番号１６で表されるアミノ酸配列からなる軽鎖可変領域を含むｓｃＦｖ、
配列番号２０で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号１７で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号１８で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号１９で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号２２で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号２４で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号２５で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号２６で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号２７で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号２８で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号２９で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
配列番号２１で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、配列番号５５で表されるアミノ酸配列の２１番目～２７１番目のアミノ酸配列からなるｓｃＦｖ、又は、
　配列番号７１で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖである、（１）～（７）のいずれか一つに記載の医薬組成物、 (7)
The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO is an scFv.
(8)
An antibody that binds to HLA/NY-ESO or an antigen-binding fragment thereof,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 30,
scFv comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29,
scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21, scFv consisting of the 21st to 271st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or
The pharmaceutical composition according to any one of (1) to (7), which is an scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71,
（９）
　ＣＤ３に結合する抗体又はその抗原結合断片が、下記（ｉ）～（Ｘ）のセットからなる群より選択される１つ又は２つ以上のセットのＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含む、（１）～（８）のいずれか一つに記載の医薬組成物：
（ｉ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
（ｉｉ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＮであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
（ｉｉｉ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＳであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
（ｉｖ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＧであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
（ｖ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＱであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
（ｖｉ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＮであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
（ｖｉｉ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＳであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３、
（ｖｉｉｉ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＡであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３、
（ｉＸ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＳであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＮであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
並びに
（Ｘ）
配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＳであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＳであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３、 (9)
The antibody or antigen-binding fragment thereof that binds to CD3 comprises one or more sets of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following sets (i) to (X), (1) ) to (8):
(i)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
(ii)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is N in the amino acid sequence represented by SEQ ID NO: 8;
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
(iii)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8;
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
(iv)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
In the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), the light chain CDRL2 consists of an amino acid sequence in which the second amino acid is G, and SEQ ID NO: 12. Light chain CDRL3 consisting of the amino acid sequence shown
(v)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
A light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), where the second amino acid is Q, and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
(vi)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
A light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), where the second amino acid is N, and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
(vii)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
A light chain CDRL2 consisting of an amino acid sequence in which the second amino acid in the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12,
(viii)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
A light chain CDRL2 consisting of an amino acid sequence in which the second amino acid is A in the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12,
(iX)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8;
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
A light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), where the second amino acid is N, and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
and (X)
Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8;
Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
A light chain CDRL2 consisting of an amino acid sequence in which the second amino acid in the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), and
Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12,
（１０）
　ＣＤ３に結合する抗体又はその抗原結合断片が、
配列番号４６で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
配列番号４７で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
配列番号５１で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
配列番号４８で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
配列番号４９で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
配列番号５０で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
配列番号５４で表されるアミノ酸配列の２７２番目～３８９番目のアミノ酸配列からなる重鎖可変領域、
配列番号５５で表されるアミノ酸配列の２７７番目～３９４番目のアミノ酸配列からなる重鎖可変領域、若しくは
配列番号５６で表されるアミノ酸配列の２７７番目～３９４番目のアミノ酸配列からなる重鎖可変領域、並びに、
配列番号４６で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
配列番号４７で表されるアミノ酸配列の１３５番目～２４１番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５１で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
配列番号４８で表されるアミノ酸配列の１３５番目～２４１番目のアミノ酸配列からなる軽鎖可変領域、
配列番号４９で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５０で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５４で表されるアミノ酸配列の４０５番目～５１１番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５５で表されるアミノ酸配列の４１０番目～５１６番目のアミノ酸配列からなる軽鎖可変領域、若しくは
配列番号５６で表されるアミノ酸配列の４１０番目～５１６番目のアミノ酸配列からなる軽鎖可変領域、
を含む、（９）に記載の医薬組成物、
（１１）
　ＣＤ３に結合する抗体又はその抗原結合断片が、
配列番号４６で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号４６で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
配列番号４７で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号４７で表されるアミノ酸配列の１３５番目～２４１番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５１で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号５１で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
配列番号４８で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号４８で表されるアミノ酸配列の１３５番目～２４１番目のアミノ酸配列からなる軽鎖可変領域、
配列番号４９で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号４９で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５０で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号５０で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５４で表されるアミノ酸配列の２７２番目～３８９番目のアミノ酸配列からなる重鎖可変領域及び配列番号５４で表されるアミノ酸配列の４０５番目～５１１番目のアミノ酸配列からなる軽鎖可変領域、
配列番号５５で表されるアミノ酸配列の２７７番目～３９４番目のアミノ酸配列からなる重鎖可変領域及び配列番号５５で表されるアミノ酸配列の４１０番目～５１６番目のアミノ酸配列からなる軽鎖可変領域、又は、
配列番号５６で表されるアミノ酸配列の２７７番目～３９４番目のアミノ酸配列からなる重鎖可変領域及び配列番号５６で表されるアミノ酸配列の４１０番目～５１６番目のアミノ酸配列からなる軽鎖可変領域、を含む、（８）又は（９）に記載の医薬組成物、 (10)
An antibody that binds to CD3 or an antigen-binding fragment thereof,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50,
A heavy chain variable region consisting of the 272nd to 389th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54,
A heavy chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or a heavy chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56. ,and,
A light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46,
A light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47,
A light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51,
A light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48,
A light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49,
A light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50,
A light chain variable region consisting of the 405th to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54,
A light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56. ,
The pharmaceutical composition according to (9), comprising:
(11)
An antibody that binds to CD3 or an antigen-binding fragment thereof,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48, and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49,
A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50,
A heavy chain variable region consisting of the 272nd to 389th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54, and a light chain variable region consisting of the 405th to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54,
A heavy chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, and a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, Or
A heavy chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, The pharmaceutical composition according to (8) or (9), comprising:
（１２）
　ＣＤ３に結合する抗体又はその抗原結合断片が、ｓｃＦｖである、（１）～（１１）のいずれか一つに記載の医薬組成物、
（１３）
　ＣＤ３に結合する抗体又はその抗原結合断片が、
配列番号４６で表されるアミノ酸配列の２番目～２４３番目のアミノ酸配列からなるｓｃＦｖ、
配列番号４７で表されるアミノ酸配列の２番目～２４１番目のアミノ酸配列からなるｓｃＦｖ、
配列番号５１で表されるアミノ酸配列の２番目～２４３番目のアミノ酸配列からなるｓｃＦｖ、
配列番号４８で表されるアミノ酸配列の２番目～２４１番目のアミノ酸配列からなるｓｃＦｖ、
配列番号４９で表されるアミノ酸配列の２番目～２４３番目のアミノ酸配列からなるｓｃＦｖ、
配列番号５０で表されるアミノ酸配列の２番目～２４３番目のアミノ酸配列からなるｓｃＦｖ、
配列番号５４で表されるアミノ酸配列の２７２番目～５１１番目のアミノ酸配列からなるｓｃＦｖ、
配列番号５５で表されるアミノ酸配列の２７７番目～５１６番目のアミノ酸配列からなるｓｃＦｖ、または
配列番号５６で表されるアミノ酸配列の２７７番目～５１６番目のアミノ酸配列からなるｓｃＦｖである、（１）～（１２）のいずれか一つに記載の医薬組成物、
（１４）
　Ｎ末端からＣ末端に向かって、ｓｃＦｖであるＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片、ｓｃＦｖであるＣＤ３に結合する抗体又はその抗原結合断片及びＦｃ領域（ｉ）をその順に含んでなる第１のポリペプチド；並びに、Ｆｃ領域（ｉｉ）を含む第２ポリペプチドを含み、好適には、Ｆｃ領域（ｉ）とＦｃ領域（ｉｉ）において会合してなる、（１）～（１３）のいずれか一つに記載の医薬組成物、
（１５）
　第１ポリペプチドが、配列番号３２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列を含む、（１４）に記載の医薬組成物、
（１６）
　第１ポリペプチドが、配列番号３２、３４、３５、３６、３７、３８、３９、４０、４１、４２、４３、３３、５２、５３もしくは５４で表されるアミノ酸配列の５２９番目～７４５番目のアミノ酸配列、又は、配列番号５５もしくは５６で表されるアミノ酸配列の５３４番目～７５０番目のアミノ酸配列を含む、（１４）又は（１５）に記載の医薬組成物、 (12)
The pharmaceutical composition according to any one of (1) to (11), wherein the antibody or antigen-binding fragment thereof that binds to CD3 is an scFv.
(13)
An antibody that binds to CD3 or an antigen-binding fragment thereof,
scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46,
scFv consisting of the 2nd to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47,
scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51,
scFv consisting of the 2nd to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48,
scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49,
scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50,
scFv consisting of the 272nd to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54,
An scFv consisting of the 277th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or an scFv consisting of the 277th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, (1) The pharmaceutical composition according to any one of ~(12),
(14)
In order from the N-terminus to the C-terminus, an antibody that binds to HLA/NY-ESO that is an scFv or an antigen-binding fragment thereof, an antibody that binds to CD3 that is an scFv or an antigen-binding fragment thereof, and an Fc region (i). and a second polypeptide comprising an Fc region (ii), preferably associated at the Fc region (i) and the Fc region (ii), (1) to ( The pharmaceutical composition according to any one of 13),
(15)
The first polypeptide is represented by the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35. The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37. , the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40. The amino acid sequence of the 21st to 511th amino acids of the amino acid sequence represented by SEQ ID NO: 41, the amino acid sequence of the 21st to 511th of the amino acid sequence represented by SEQ ID NO: 42, the amino acid sequence of the 21st to 511th amino acids of the amino acid sequence represented by SEQ ID NO: 42, The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, Amino acid sequence, amino acid sequence from 21st to 511th of the amino acid sequence represented by SEQ ID NO: 53, amino acid sequence from 21st to 511th of the amino acid sequence represented by SEQ ID NO: 54, amino acid sequence represented by SEQ ID NO: 55 The pharmaceutical composition according to (14), comprising the 21st to 516th amino acid sequence of or the 21st to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56,
(16)
The first polypeptide is located at positions 529 to 745 of the amino acid sequence represented by SEQ ID NO: 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 33, 52, 53 or The pharmaceutical composition according to (14) or (15), comprising the amino acid sequence or the 534th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55 or 56,
（１７）
　第１ポリペプチドが、配列番号３２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、列番号５５で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列からなる、（１４）～（１６）のいずれか一つに記載の医薬組成物、
（１８）
　第２ポリペプチドが、配列番号３１で示されるアミノ酸配列の２０番目～２４６番目のアミノ酸配列を含んでなる、（１４）～（１７）のいずれか一つに記載の医薬組成物、
（１９）
　第１ポリペプチド、第２ポリペプチド及び、第３ポリペプチドを含み、
第１ポリペプチドが、Ｎ末端からＣ末端に向かって、ｓｃＦｖであるＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片、ｓｃＦｖであるＣＤ３に特異的に結合する抗体又はその抗原結合断片及びＦｃ領域（ｉ）を、その順に含んでなり、
　第２ポリペプチドが、Ｆｃ領域（ｉｉ）を含む免疫グロブリン重鎖からなり、
　第３ポリペプチドが免疫グロブリン軽鎖からなり、
　好適には、第２ポリペプチドと第３ポリペプチドが会合しており、
　第１ポリペプチドと第２ペプチドが、それぞれのＦｃ領域において会合している、（１）～（１３）のいずれか一つに記載の医薬組成物、
（２０）
　第２ポリペプチドが、配列番号４４で表されるアミノ酸配列のアミノ酸番号２０～２４２のアミノ酸配列を含む、（１９）に記載の医薬組成物、
（２１）
　第３ポリペプチドが配列番号４５で表されるアミノ酸配列を含む、（１９）又は（２０）に記載の医薬組成物、
（２２）
　第１ポリペプチドが、配列番号３２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列を含む、（１９）～（２１）のいずれか一つに記載の医薬組成物、
（２３）
　第１ポリペプチドが、配列番号３２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列及び配列番号３３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列からなる、（１９）～（２２）のいずれか一つに記載の医薬組成物、 (17)
The first polypeptide is represented by the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35. The 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37. , the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40. Amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: 41, amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: 42, amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: The 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, Amino acid sequence, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence represented by SEQ ID NO: 55 or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, the pharmaceutical composition according to any one of (14) to (16). thing,
(18)
The pharmaceutical composition according to any one of (14) to (17), wherein the second polypeptide comprises the 20th to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 31;
(19)
comprising a first polypeptide, a second polypeptide, and a third polypeptide,
The first polypeptide includes, from the N-terminus to the C-terminus, an antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO, which is an scFv, an antibody or antigen-binding fragment thereof, which specifically binds to CD3, which is an scFv; Fc region (i) in that order,
the second polypeptide consists of an immunoglobulin heavy chain comprising an Fc region (ii);
the third polypeptide consists of an immunoglobulin light chain,
Preferably, the second polypeptide and the third polypeptide are associated,
The pharmaceutical composition according to any one of (1) to (13), wherein the first polypeptide and the second peptide are associated in their respective Fc regions;
(20)
The pharmaceutical composition according to (19), wherein the second polypeptide comprises the amino acid sequence of amino acids 20 to 242 of the amino acid sequence represented by SEQ ID NO: 44,
(21)
The pharmaceutical composition according to (19) or (20), wherein the third polypeptide comprises the amino acid sequence represented by SEQ ID NO: 45,
(22)
The first polypeptide is represented by the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35. The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37. , the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40. The amino acid sequence of the 21st to 511th amino acids of the amino acid sequence represented by SEQ ID NO: 41, the amino acid sequence of the 21st to 511th of the amino acid sequence represented by SEQ ID NO: 42, the amino acid sequence of the 21st to 511th amino acids of the amino acid sequence represented by SEQ ID NO: 42, The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, Amino acid sequence, amino acid sequence from 21st to 511th of the amino acid sequence represented by SEQ ID NO: 53, amino acid sequence from 21st to 511th of the amino acid sequence represented by SEQ ID NO: 54, amino acid sequence represented by SEQ ID NO: 55 or the 21st to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, the pharmaceutical composition according to any one of (19) to (21). thing,
(23)
The first polypeptide is represented by the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35. The 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37. , the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40. Amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: 41, amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: 42, amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: The 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52. Amino acid sequence, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence represented by SEQ ID NO: 55 or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, the pharmaceutical composition according to any one of (19) to (22). thing,
（２４）
　Ｎ末端からＣ末端に向かって、
ｓｖＦｖであるＨＬＡ／ＮＹ－ＥＳＯに特異的に結合する抗体又はその抗原結合断片、ＣＤ３に結合する抗体の重鎖可変領域及び定常領域ＣＨ１並びに免疫グロブリンＦｃ領域（ｉ）をその順で含んでなる第１ポリペプチド、
免疫グロブリンのヒンジ領域及びＦｃ領域（ｉｉ）を含んでなる第２ポリペプチド、並びに、
可変領域及び定常領域からなるＣＤ３に結合する抗体軽鎖を含む第３ポリペプチドを含み、
好適には、第１ポリペプチドと第２ポリペプチドがＦｃ領域（ｉ）とＦｃ領域（ｉｉ）において会合しており、第１ポリペプチドが抗体重鎖の可変領域及び定常領域ＣＨ１において第３ポリペプチドと会合してなる、（９）～（１１）のいずれか一つに記載の医薬組成物、
（２５）
　第１ポリペプチドが、配列番号５７で表されるアミノ酸配列の２０番目～７２４番目のアミノ酸配列、配列番号６０で表されるアミノ酸配列の２０番目～７１９番目のアミノ酸配、又は、配列番号６１で表されるアミノ酸配列２０番目～７１９番目のアミノ酸配列を含む、（９）～（１１）及び（２４）のいずれか一つに記載の医薬組成物、
（２６）
　第２ポリペプチドが、配列番号３１で示されるアミノ酸配列の２０番目～２４６番目のアミノ酸配列を含んでなる、（９）～（１１）、（２４）及び（２５）のいずれか一つに記載の医薬組成物、
（２７）
　第３ポリペプチドが、配列番号５８で表されるアミノ酸配列の２１番目～１２７番目のアミノ酸配列を含んでなる、（９）～（１１）及び（２４）～（２６）のいずれか一つに記載の医薬組成物、
（２８）
　第３ポリペプチドが、配列番号５８で表されるアミノ酸配列の２１番目～２３３番目のアミノ酸配列を含んでなる、（９）～（１１）及び（２４）～（２７）のいずれか一つに記載の医薬組成物、
（２９）
　（１）～（２８）のいずれか一つに記載の医薬組成物であって、該医薬組成物に含まれる１つ又は２つ以上のポリペプチドの有するアミノ酸配列において、該配列のカルボキシル末端から１つ又は２つのアミノ酸が欠失してなる、該医薬組成物、
（３０）
　がんが、腎がん、メラノーマ、有棘細胞がん、基底細胞がん、結膜がん、口腔がん、喉頭がん、咽頭がん、甲状腺がん、肺がん（非小細胞肺がん（腺がん、扁平上皮がん、大細胞がん）、小細胞肺がん）、乳がん、食道がん、胃がん、十二指腸がん、小腸がん、大腸がん、直腸がん、虫垂がん、肛門がん、肝がん、胆嚢がん、胆管がん、膵がん、副腎がん、膀胱がん、前立腺がん、子宮がん、膣がん、脂肪肉腫、血管肉腫、軟骨肉腫、横紋筋肉腫、ユーイング肉腫、骨肉腫、未分化多型肉腫、粘液型線維肉腫、悪性末梢性神経鞘腫、後腹膜肉腫、滑膜肉腫、子宮肉腫、消化管間質腫瘍、平滑筋肉腫、類上皮肉腫、Ｂ細胞リンパ腫、Ｔ・ＮＫ細胞リンパ腫、ホジキンリンパ腫、骨髄性白血病、リンパ性白血病、骨髄増殖性疾患、骨髄異形成症候群、多発性骨髄腫、精巣がん及び卵巣がんからなる群から選択される１つ又は２つ以上である、（１）～（２９）のいずれか一つに記載の医薬組成物、 (24)
From the N-terminus to the C-terminus,
An antibody that specifically binds to HLA/NY-ESO, which is svFv, or an antigen-binding fragment thereof, comprising the heavy chain variable region and constant region CH1 of an antibody that binds to CD3, and immunoglobulin Fc region (i) in that order. a first polypeptide,
a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii); and
comprising a third polypeptide comprising an antibody light chain that binds to CD3, consisting of a variable region and a constant region,
Preferably, the first polypeptide and the second polypeptide are associated in Fc region (i) and Fc region (ii), and the first polypeptide is associated with a third polypeptide in the variable region and constant region CH1 of the antibody heavy chain. The pharmaceutical composition according to any one of (9) to (11), which is associated with a peptide;
(25)
The first polypeptide is the 20th to 724th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 57, the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 60, or the amino acid sequence of SEQ ID NO: 61. The pharmaceutical composition according to any one of (9) to (11) and (24), comprising the amino acid sequence 20th to 719th of the represented amino acid sequence,
(26)
According to any one of (9) to (11), (24) and (25), the second polypeptide comprises the 20th to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 31. a pharmaceutical composition of
(27)
Any one of (9) to (11) and (24) to (26), wherein the third polypeptide comprises the 21st to 127th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 58. The pharmaceutical composition described,
(28)
Any one of (9) to (11) and (24) to (27), wherein the third polypeptide comprises the 21st to 233rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 58. The pharmaceutical composition described,
(29)
The pharmaceutical composition according to any one of (1) to (28), wherein in the amino acid sequence of one or more polypeptides contained in the pharmaceutical composition, from the carboxyl terminus of the sequence The pharmaceutical composition, which has one or two amino acids deleted;
(30)
Cancer: renal cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cavity cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer (non-small cell lung cancer) cancer, squamous cell carcinoma, large cell carcinoma), small cell lung cancer), breast cancer, esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, colon cancer, rectal cancer, appendiceal cancer, anal cancer, Liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal gland cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, B 1 selected from the group consisting of cell lymphoma, T/NK cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, testicular cancer, and ovarian cancer. the pharmaceutical composition according to any one of (1) to (29), which is one or more;
（３１）
　免疫チェックポイント分子を阻害する化合物又は化学療法剤の後に投与される、（１）～（３０）のいずれか一つに記載の医薬組成物。
（３２）
　免疫チェックポイント分子を阻害する化合物又は化学療法剤より前に投与される、（１）～（３０）のいずれか一つに記載の医薬組成物、
（３３）
　免疫チェックポイント分子を阻害する化合物又は化学療法剤と同時に投与される、（１）～（３０）のいずれか一つに記載の医薬組成物、
（３４）
　免疫チェックポイント分子を阻害する化合物又は化学療法剤を含む、（１）～（３０）及び（３３）のいずれか一つに記載の医薬組成物、
（３５）
　さらなる１つ又は２つ以上の医薬及び／又は治療と組み合わせて使用される、（１）～（３４）のいずれか一つに記載の医薬組成物、
（３６）
　［ｉｉ］記載の化合物が免疫チェックポイント分子を阻害する化合物である、（１）～（３５）のいずれか一つに記載の医薬組成物、
（３７）
　免疫チェックポイント分子がＰＤ－１、ＰＤ－Ｌ１、ＰＤ－Ｌ２、ＣＴＬＡ－４及びＴＩＧＩＴよりなる群から選択される、（１）～（３６）のいずれか一つに記載の医薬組成物、
（３８）
　免疫チェックポイント分子がＰＤ－１である、（１）～（３７）のいずれか一つに記載の医薬組成物、
（３９）
　免疫チェックポイント分子を阻害する化合物がニボルマブ若しくはペムブロリズマブ、その抗原結合断片、又は、その抗原結合断片を含む化合物である、（３８）記載の医薬組成物。
（４０）
　免疫チェックポイント分子がＰＤ－Ｌ１である、（１）～（３７）のいずれか一つに記載の医薬組成物、
（４１）
　免疫チェックポイント分子を阻害する化合物がアテゾリズマブ、デュルバルマブ若しくはアベルマブ、その抗原結合断片、又は、その抗原結合断片を含む化合物である、（４０）記載の医薬組成物、
（４２）
　免疫チェックポイント分子がＣＴＬＡ－４である、（１）～（３７）のいずれか一つに記載の医薬組成物、
（４３）
　免疫チェックポイント分子を阻害する化合物がイピリムマブ、トレメリムマブ、スパルタリズマブ若しくはセミプリマブ、その抗原結合断片、又は、その抗原結合断片を含む化合物である、（４２）記載の医薬組成物、
（４４）
　免疫チェックポイント分子がＴＩＧＩＴである、（１）～（３７）のいずれか一つに記載の医薬組成物、
（４５）
　免疫チェックポイント分子を阻害する化合物がチラゴルマブ若しくはビボストリマブ、その抗原結合断片、又は、その抗原結合断片を含む化合物である、（４４）記載の医薬組成物、
（４６）
　免疫チェックポイント分子を阻害する化合物がペムブロリズマブ、ニボルマブ、スパルタリズマブ、セミプリマブ、アベルマブ、アテゾリズマブ、ヂュルバルマブ、イピリムマブ、又は、トレメリムマブである、（１）～（３６）のいずれか一つに記載の医薬組成物、
（４７）
　免疫チェックポイント分子を阻害する化合物が、ペムブロリズマブ若しくはペムブロリズマブの抗原結合断片、又は、その抗原結合断片を含む化合物である、（１）～（３９）のいずれか一つに記載の医薬組成物、
（４８）
　［ｉｉ］記載の化合物が化学療法剤である、（１）～（３５）のいずれか一つに記載の医薬組成物、
（４９）
　化学療法剤が微小管形成阻害剤である、（１）～（３５）及び（４８）のいずれか一つに記載の医薬組成物、 (31)
The pharmaceutical composition according to any one of (1) to (30), which is administered after a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule.
(32)
The pharmaceutical composition according to any one of (1) to (30), which is administered before a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule,
(33)
The pharmaceutical composition according to any one of (1) to (30), which is administered simultaneously with a compound that inhibits an immune checkpoint molecule or a chemotherapeutic agent;
(34)
The pharmaceutical composition according to any one of (1) to (30) and (33), comprising a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule,
(35)
The pharmaceutical composition according to any one of (1) to (34), used in combination with one or more additional medicaments and/or treatments,
(36)
[ii] The pharmaceutical composition according to any one of (1) to (35), wherein the compound described above is a compound that inhibits an immune checkpoint molecule;
(37)
The pharmaceutical composition according to any one of (1) to (36), wherein the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4 and TIGIT,
(38)
The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is PD-1;
(39)
The pharmaceutical composition according to (38), wherein the compound that inhibits an immune checkpoint molecule is nivolumab or pembrolizumab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof.
(40)
The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is PD-L1;
(41)
The pharmaceutical composition according to (40), wherein the compound that inhibits an immune checkpoint molecule is atezolizumab, durvalumab, or avelumab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof;
(42)
The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is CTLA-4;
(43)
The pharmaceutical composition according to (42), wherein the compound that inhibits an immune checkpoint molecule is ipilimumab, tremelimumab, spartalizumab, or cemiplimab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof;
(44)
The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is TIGIT;
(45)
The pharmaceutical composition according to (44), wherein the compound that inhibits an immune checkpoint molecule is tiragolumab or vivostrimab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof;
(46)
The pharmaceutical composition according to any one of (1) to (36), wherein the compound that inhibits immune checkpoint molecules is pembrolizumab, nivolumab, spartalizumab, cemiplimab, avelumab, atezolizumab, dulvalumab, ipilimumab, or tremelimumab. thing,
(47)
The pharmaceutical composition according to any one of (1) to (39), wherein the compound that inhibits an immune checkpoint molecule is pembrolizumab, an antigen-binding fragment of pembrolizumab, or a compound containing an antigen-binding fragment thereof;
(48)
[ii] The pharmaceutical composition according to any one of (1) to (35), wherein the compound described above is a chemotherapeutic agent;
(49)
The pharmaceutical composition according to any one of (1) to (35) and (48), wherein the chemotherapeutic agent is a microtubule formation inhibitor;
（５０）
　化学療法剤がタキサン系の微小管形成阻害剤である、（１）～（３５）、（４８）及び（４９）のいずれか一つに記載の医薬組成物、
（５１）
　化学療法剤がパクリタキセル、ドセタキセル、ビンクリスチン、ビンブラスチン、ビンデシン、エリブリン、ビノレルビン、アルブミン懸濁型パクリタキセルオキサリプラチン、カルボプラチン、シスプラチン、ネダプラチン、アザシチジン、デシタビン、ドキソルビシン、ブレオマイシン、リポソーマルドキソルビシン、イホスファミド、シクロホスファミド、及び、ダカルバジンよりなる群から選択される、（１）～（３５）及び（４８）～（５０）のいずれか一つに記載の医薬組成物、
（５２）
　化学療法剤がパクリタキセル又はアルブミン懸濁型パクリタキセルである、（１）～（３５）及び（４８）～（５１）のいずれか一つに記載の医薬組成物、
（５３）
　さらにマルチキナーゼ阻害剤と組み合わせて使用される、（１）～（５２）のいずれか一つに記載の医薬組成物、
（５４）
　マルチキナーゼ阻害剤がソラフェニブ、スニチニブ、パゾパニブ、レゴラフェニブ、及びレンバチニブよりなる群から選択される１つ又は２つ以上である、（５３）に記載の医薬組成物、
（５５）
　マルチキナーゼ阻害剤が、レンバチニブである、（５３）又は（５４）記載の医薬組成物、
（５６）
　［ｉｉ］記載の化合物が、免疫チェックポイント分子を阻害する化合物であり、好適には、ペムブロリズマブ若しくはペムブロリズマブの抗原結合断片、又はその抗原結合断片を含む化合物である、（５３）～（５５）のいずれか一つに記載の医薬組成物、
（５７）
　免疫チェックポイント分子を阻害する化合物が、ペムブロリズマブ若しくはペムブロリズマブの抗原結合断片、又はその抗原結合断片を含む化合物であり、マルチキナーゼ阻害剤が、レンバチニブである、（５３）～（５６）のいずれか一つに記載の医薬組成物、
（５８）
　化学療法剤がパクリタキセル、ドセタキセル、ビンクリスチン、ビンブラスチン、ビンデシン、エリブリン、ビノレルビン及びアルブミン懸濁型パクリタキセルよりなる群から選択されるタキサン系の微小管形成阻害剤である、（１）～（３５）及び（４８）～（５２）のいずれか一つに記載の医薬組成物、
（５９）
　がんの治療及び／又は予防のための、（１）～（４７）のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤、
（６０）
　がんの治療及び／又は予防のための、（５３）～（５７）のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤、並びに、マルチキナーゼ阻害剤、
（６１）
　がんの治療及び／又は予防のための、（１）～（４７）のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤、
（６２）
　がんの治療及び／又は予防のための医薬組成物を調製するための、（５３）～（５７）のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤、並びに、マルチキナーゼ阻害剤の使用、
（６３）
　（１）～（４７）のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤を投与することを含む、がんを治療及び／又は予防する方法、
（６４）
　（５３）～（５７）のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤、並びに、マルチキナーゼ阻害剤を投与することを含む、がんを治療及び／又は予防する方法、
等に関する。 (50)
The pharmaceutical composition according to any one of (1) to (35), (48) and (49), wherein the chemotherapeutic agent is a taxane-based microtubule formation inhibitor;
(51)
Chemotherapy agents include paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, albumin suspension paclitaxel oxaliplatin, carboplatin, cisplatin, nedaplatin, azacitidine, decitabine, doxorubicin, bleomycin, liposomal doxorubicin, ifosfamide, cyclophosphamide and dacarbazine, the pharmaceutical composition according to any one of (1) to (35) and (48) to (50),
(52)
The pharmaceutical composition according to any one of (1) to (35) and (48) to (51), wherein the chemotherapeutic agent is paclitaxel or albumin-suspended paclitaxel;
(53)
The pharmaceutical composition according to any one of (1) to (52), which is further used in combination with a multikinase inhibitor;
(54)
The pharmaceutical composition according to (53), wherein the multikinase inhibitor is one or more selected from the group consisting of sorafenib, sunitinib, pazopanib, regorafenib, and lenvatinib;
(55)
The pharmaceutical composition according to (53) or (54), wherein the multikinase inhibitor is lenvatinib;
(56)
[ii] The compound described in (53) to (55) is a compound that inhibits an immune checkpoint molecule, and is preferably a compound containing pembrolizumab or an antigen-binding fragment of pembrolizumab, or an antigen-binding fragment thereof. The pharmaceutical composition according to any one of
(57)
Any one of (53) to (56), wherein the compound that inhibits an immune checkpoint molecule is pembrolizumab, an antigen-binding fragment of pembrolizumab, or a compound containing an antigen-binding fragment thereof, and the multikinase inhibitor is lenvatinib. The pharmaceutical composition described in
(58)
(1) to (35), wherein the chemotherapeutic agent is a taxane-based microtubule formation inhibitor selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, and albumin-suspended paclitaxel; The pharmaceutical composition according to any one of 48) to (52),
(59)
A compound or chemotherapeutic agent that inhibits [i] the multispecific antibody and [ii] immune checkpoint molecules according to any one of (1) to (47) for the treatment and/or prevention of cancer. ,
(60)
A compound or chemotherapeutic agent that inhibits [i] the multispecific antibody and [ii] immune checkpoint molecules according to any one of (53) to (57) for the treatment and/or prevention of cancer. , as well as multi-kinase inhibitors,
(61)
A compound or chemotherapeutic agent that inhibits [i] the multispecific antibody and [ii] immune checkpoint molecules according to any one of (1) to (47) for the treatment and/or prevention of cancer. ,
(62)
[i] Multispecific antibody and [ii] immune checkpoint molecule according to any one of (53) to (57) for preparing a pharmaceutical composition for treating and/or preventing cancer. compounds or chemotherapeutic agents that inhibit , as well as the use of multikinase inhibitors,
(63)
Treating and treating cancer, comprising administering [i] the multispecific antibody according to any one of (1) to (47) and [ii] a compound or chemotherapeutic agent that inhibits immune checkpoint molecules. /or a method of preventing;
(64)
(53) to administering the multispecific antibody and [ii] a compound or chemotherapeutic agent that inhibits immune checkpoint molecules, and a multikinase inhibitor according to any one of (53) to (57). A method of treating and/or preventing cancer, including
Regarding etc.
　本発明の提供する抗体および他剤の組合せを用いることにより各種癌の治療または予防が可能となる。 By using the combination of the antibody provided by the present invention and other drugs, it is possible to treat or prevent various cancers.
図1（１）は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｐａｃｌｉｔａｘｅｌ単独投与、及びＮＹＺ－１０１０とＰａｃｌｉｔａｘｅｌの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 1 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Paclitaxel alone, and NYZ-1010 and Paclitaxel in a human T cell transfer model. FIG. 3 is a diagram showing the antitumor activity in combination administration of . Error bars in the figure indicate standard error (n=5). 図１（２）はヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｐａｃｌｉｔａｘｅｌ単独投与、及びＮＹＺ－１０１０とＰａｃｌｉｔａｘｅｌの併用投与でのＤａｙ１２の腫瘍体積をベースラインとした各個体のＤａｙ３１の腫瘍体積変化率を示した図である。Figure 1 (2) shows the administration of Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Paclitaxel alone, and NYZ-1010 and Paclitaxel in a human T cell transfer model. It is a figure showing the tumor volume change rate on Day 31 of each individual with the tumor volume on Day 12 as the baseline in combination administration. 図２（１）は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｎａｂ　Ｐａｃｌｉｔａｘｅｌ単独投与、及びＮＹＺ－１０１０とＮａｂ Ｐａｃｌｉｔａｘｅｌの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 2 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Nab Paclitaxel alone, and NYZ-1010 and NYZ-1010 in a human T cell transfer model. FIG. 2 is a diagram showing the antitumor activity of combined administration of Nab Paclitaxel. Error bars in the figure indicate standard error (n=5). 図２（２）はヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｎａｂ Ｐａｃｌｉｔａｘｅｌ単独投与、及びＮＹＺ－１０１０とＮａｂ Ｐａｃｌｉｔａｘｅｌの併用投与でのＤａｙ１３の腫瘍体積をベースラインとした各個体のＤａｙ３８の腫瘍体積変化率を示した図である。Figure 2 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Nab Paclitaxel alone, and NYZ-1010 and Nab in a human T cell transfer model. It is a figure showing the tumor volume change rate on Day 38 of each individual with the tumor volume on Day 13 as the baseline in combination administration of Paclitaxel. 図３は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｐｅｍｂｒｏｌｉｚｕｍａｂ単独投与、及びＮＹＺ－１０１０とＰｅｍｂｒｏｌｉｚｕｍａｂの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 3 shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Pembrolizumab alone, and NYZ-1010 and Pembrolizumab combined in a human T cell transfer model. FIG. 2 is a diagram showing the antitumor activity of Error bars in the figure indicate standard error (n=5). 図４は、ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＦ－００１６単独投与、抗マウスＰＤ－１抗体単独投与、及びＮＹＦ－００１６と抗マウスＰＤ－１抗体の併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 4 shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYF-0016 alone, anti-mouse PD-1 antibody alone, and NYF-0016 in a human CD3ε knock-in mouse model. FIG. 2 is a diagram showing the antitumor activity of the combined administration of anti-mouse PD-1 antibody and anti-mouse PD-1 antibody. Error bars in the figure indicate standard error (n=5). 本発明に示される抗体のフォーマットを示した図。(5a) ｓｃＦｖ：抗体Ｈ鎖可変領域（ＶＨ、後述）及び抗体Ｌ鎖可変領域（ＶＬ、後述）（いずれも白）をリンカーで繋げたフォーマットである。本発明の実施例では抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ及びＣＤ３のｓｃＦｖを評価に用いた。(5b) Ｆａｂ：ＶＨ（白）と抗体Ｈ鎖定常領域(ＣＨ１：市松模様)及びＶＬ（白）と抗体Ｌ鎖定常領域(横線)からなるフォーマットである。本発明の実施例では抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　Ｆａｂ等を評価に用いた。(5c) ｔａＦｖ：２種類のｓｃＦｖ（白と右上斜線）をリンカーでつなげたフォーマットである。本発明の実施例では抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖと抗ＣＤ３　ｓｃＦｖを含むｔａＦｖを評価に用いた。(5d) ｔａＦｖ－ヘテロダイマーＦｃ型：ｔａＦｖのＣ末端側にヘテロダイマーを形成する変異をいれたＦｃ（左上斜線）が付加し（第１ポリペプチドともいう）、もう一つのＦｃ（黒塗り：第２ポリペプチドともいう）とヘテロに会合したフォーマットである。本発明の実施例では抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖと抗ＣＤ３　ｓｃＦｖを含むｔａＦｖ－ヘテロダイマーＦｃを評価に用いた。(5e) ｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型：上記ｔａＦｖ－ヘテロダイマーＦｃ型にＦａｂを付加したフォーマットである。本発明の実施例では抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖと抗ＣＤ３　ｓｃＦｖを含むｔａＦｖ及びＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　Ｆａｂを用いたｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ等を評価に用いた。FIG. 2 is a diagram showing the format of antibodies shown in the present invention. (5a) scFv: A format in which an antibody H chain variable region (VH, described below) and an antibody L chain variable region (VL, described below) (both white) are connected with a linker. In the examples of the present invention, anti-HLA-A2/NY-ESO and CD3 scFv were used for evaluation. (5b) Fab: A format consisting of VH (white) and antibody H chain constant region (CH1: checkered pattern) and VL (white) and antibody L chain constant region (horizontal line). In the examples of the present invention, anti-HLA-A2/NY-ESO Fab etc. were used for evaluation. (5c) taFv: A format in which two types of scFv (white and upper right diagonal line) are connected by a linker. In the examples of the present invention, taFv including anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv were used for evaluation. (5d) taFv-heterodimer Fc type: Fc with a mutation that forms a heterodimer (hatched line on the upper left) is added to the C-terminal side of taFv (also called the first polypeptide), and another Fc (blacked out: (also referred to as a second polypeptide). In Examples of the present invention, taFv-heterodimer Fc containing anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv was used for evaluation. (5e) taFv-Fab-heterodimer Fc type: This is a format in which Fab is added to the above taFv-heterodimer Fc type. In the examples of the present invention, taFv containing anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv, and taFv-Fab-heterodimer Fc using HLA-A2/NY-ESO Fab were used for evaluation. 本発明に示される抗体のフォーマットを示した図。(6a) ｔａＦｖ－ヘテロダイマーＦｃ型及びｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型に共通である第１ポリペプチドを示す。第１ポリペプチドは、Ｎ末端からＣ末端に向かって、ヒトＨＬＡ／ＮＹ－ＥＳＯに特異的に結合するｓｃＦｖ、ＣＤ３に特異的に結合するｓｃＦｖ及びＦｃ領域（ｉ）を、その順に含んでなる。(6b) ｔａＦｖ－ヘテロダイマーＦｃ型の第２ポリペプチドを示す。第２ポリペプチドは、ヒンジ領域及びＦｃ領域（ｉｉ）を含んでなる。(6c) ｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型の第２ポリペプチドを示す。第２ポリペプチドは、ヒンジ領域及びＦｃ領域（ｉｉ）を含む免疫グロブリン重鎖を含んでなる。(6d) ｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型の第３ポリペプチドを示す。第３ポリペプチドは、免疫グロブリン軽鎖を含んでなる。FIG. 2 is a diagram showing the format of antibodies shown in the present invention. (6a) Shows the first polypeptide common to taFv-heterodimer Fc type and taFv-Fab-heterodimer Fc type. The first polypeptide comprises, in order from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA/NY-ESO, an scFv that specifically binds to CD3, and an Fc region (i). . (6b) Shows the second polypeptide of taFv-heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii). (6c) Shows the second polypeptide of taFv-Fab-heterodimer Fc type. The second polypeptide comprises an immunoglobulin heavy chain including a hinge region and an Fc region (ii). (6d) Shows the third polypeptide of taFv-Fab-heterodimer Fc type. The third polypeptide comprises an immunoglobulin light chain. 本発明に含まれる抗体のフォーマットを示した図。(７a) Ｈｙｂrｉｄ型：Ｆａｂ及びｓｃＦｖそれぞれのＣ末端側にへテロダイマーを形成する変異をいれたＦｃ（斜線及び黒塗り）が付加し、２つのＦｃが会合したフォーマットである。本発明の実施例では抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　Ｆａｂと抗ＣＤ３　ｓｃＦｖを含むＨｙｂrｉｄ型を評価に用いた。(７b) Ｄｕａｌ型：２種類の異なるｓｃＦｖそれぞれのＣ末端側にへテロダイマーを形成する変異をいれたＦｃ（左上斜線及び黒塗り）が付加し、２つのＦｃヘテロに会合したフォーマットである。本発明の実施例では抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖと抗ＣＤ３　ｓｃＦｖを含むＤｕａｌ型を評価に用いた。(７c) ｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型：ｓｃＦｖとＦａｂをリンカーで繋げたＣ末端側にヘテロダイマーを形成する変異をいれたＦｃ（左上斜線）が付加し、もう１つのＦｃ（黒塗り）と会合したフォーマットである。本発明の実施例では抗ＣＤ３　ｓｃＦｖ（右上斜線）と抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　Ｆａｂを含むｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型を評価に用いた。また、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖ（右上斜線）と抗ＣＤ３　Ｆａｂを含むｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型も評価に用いた。FIG. 2 is a diagram showing the format of antibodies included in the present invention. (7a) Hybrid type: This is a format in which an Fc containing a mutation that forms a heterodimer (hatched and black) is added to the C-terminal side of each of Fab and scFv, and the two Fcs are associated. In the examples of the present invention, a hybrid type containing anti-HLA-A2/NY-ESO Fab and anti-CD3 scFv was used for evaluation. (7b) Dual type: This is a format in which an Fc containing a mutation that forms a heterodimer (upper left diagonal line and black) is added to the C-terminal side of each of two different scFvs, and the two Fc heteros are associated. In the examples of the present invention, a dual type containing anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv was used for evaluation. (7c) scFv-Fab-heterodimer Fc type: An Fc with a mutation to form a heterodimer (upper left diagonal line) is added to the C-terminus of the scFv and Fab connected with a linker, and another Fc (blacked out) This is the format in which we met. In the examples of the present invention, scFv-Fab-heterodimer Fc type containing anti-CD3 scFv (upper right diagonal line) and anti-HLA-A2/NY-ESO Fab was used for evaluation. In addition, an scFv-Fab-heterodimer Fc type containing anti-HLA-A2/NY-ESO scFv (upper right diagonal line) and anti-CD3 Fab was also used for evaluation. 本発明に示される抗体のフォーマットを示した図。(8a) ｔａＦｖ－ヘテロダイマーＦｃ型：図５(5d)に同じ。ｔａＦｖのＣ末端側にヘテロダイマーを形成する変異をいれたＦｃ（左上斜線）が付加し、もう１つのＦｃ（黒塗り）とヘテロに会合したフォーマットである。本発明の実施例では抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖと抗ＣＤ３　ｓｃＦｖからなるｔａＦｖ－ヘテロダイマーＦｃ型を評価に用いた。(8b) ｔａＦｖ（ｉｎｖｅｒｓｅｄ）－ヘテロダイマーＦｃ型：上記ｔａＦｖ－ヘテロダイマーＦｃ型における２つのｓｃＦｖの順序を入れかえたもの。FIG. 2 is a diagram showing the format of antibodies shown in the present invention. (8a) taFv-heterodimer Fc type: Same as Figure 5(5d). This is a format in which an Fc containing a mutation that forms a heterodimer (shaded in the upper left) is added to the C-terminal side of taFv, and it is heterozygously associated with another Fc (black). In the examples of the present invention, a taFv-heterodimer Fc type consisting of anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv was used for evaluation. (8b) taFv (inverted)-heterodimer Fc type: the above taFv-heterodimer Fc type in which the order of the two scFvs is reversed. 本発明に示される抗体のフォーマットを示した図。(9a) ｔａＦｖ（ｉｎｖｅｒｓｅｄ）－ヘテロダイマーＦｃ型の第１ポリペプチドを示す。第１ポリペプチドは、Ｎ末端からＣ末端に向かって、ＣＤ３に特異的に結合するｓｃＦｖ、ヒトＨＬＡ／ＮＹ－ＥＳＯに特異的に結合するｓｃＦｖ及びＦｃ領域（ｉ）をその順に含んでなる。(9b) ｔａＦｖ（ｉｎｖｅｒｓｅｄ）－ヘテロダイマーＦｃ型の第２ポリペプチドを示す。第２ポリペプチドは、ヒンジ領域及びＦｃ領域（ｉｉ）を含んでなる。FIG. 2 is a diagram showing the format of antibodies shown in the present invention. (9a) taFv (inverted) - shows the first polypeptide of the heterodimer Fc type. The first polypeptide comprises, in order from the N-terminus to the C-terminus, an scFv that specifically binds to CD3, an scFv that specifically binds to human HLA/NY-ESO, and an Fc region (i). (9b) taFv (inverted) - shows a second polypeptide of the heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii). ヒトＣＤ３εのアミノ酸配列。ＮＣＢＩ／ＧｅｎＰｅｐｔにアクセッション番号：ＮＰ＿０００７２４（ＮＭ＿０００７２４．１）で登録されている。Amino acid sequence of human CD3ε. It is registered in NCBI/GenPept with accession number: NP_000724 (NM_000724.1). ＮＹＡ－０００１に含まれるＣＤＲＨ１～３及びＣＤＲＬ１～３のアミノ酸配列（配列番号１～４及び６。５番目はＤＮＮであり図８２の配列番号５）。Amino acid sequences of CDRH1-3 and CDRL1-3 contained in NYA-0001 (SEQ ID NO: 1-4 and 6. The fifth is DNN, SEQ ID NO: 5 in Figure 82). Ｃ３Ｅ－７０８５の重鎖ＣＤＲH１～ＣＤＲH３及び軽鎖ＣＤＲL１～ＣＤＲL３のアミノ酸配列（配列番号７～１０及び１２。１１番目はＲＤＤであり図８２の配列番号１１）。Amino acid sequences of heavy chain CDRH1 to CDRH3 and light chain CDRL1 to CDRL3 of C3E-7085 (SEQ ID NO: 7 to 10 and 12; position 11 is RDD; SEQ ID NO: 11 in Figure 82). ＮＹＡ－０００１の重鎖可変領域のアミノ酸配列（配列番号１３）。Amino acid sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO: 13). ＮＹＡ－０００１の軽鎖可変領域のアミノ酸配列（配列番号１４）。Amino acid sequence of the light chain variable region of NYA-0001 (SEQ ID NO: 14). ＮＹＡ－００８２の重鎖可変領域のアミノ酸配列（配列番号１５）。Amino acid sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO: 15). ＮＹＡ－００８２の軽鎖可変領域のアミノ酸配列（配列番号１６）。Amino acid sequence of the light chain variable region of NYA-0082 (SEQ ID NO: 16). ＮＹＡ－１１６３全長のアミノ酸配列（配列番号１７）。シグナル配列（１－１９）、ＮＹＡ－１１６３（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-1163 full-length amino acid sequence (SEQ ID NO: 17). Signal sequence (1-19), NYA-1163 (21-266), FLAG-His tag (267-292). ＮＹＡ－２０２３全長のアミノ酸配列（配列番号１８）。シグナル配列（１－１９）、ＮＹＡ－２０２３（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2023 full-length amino acid sequence (SEQ ID NO: 18). Signal sequence (1-19), NYA-2023 (21-266), FLAG-His tag (267-292). ＮＹＡ－２０２７全長のアミノ酸配列（配列番号１９）。シグナル配列（１－１９）、ＮＹＡ－２０２７（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2027 full-length amino acid sequence (SEQ ID NO: 19). Signal sequence (1-19), NYA-2027 (21-266), FLAG-His tag (267-292). ＮＹＡ－１１４３全長のアミノ酸配列（配列番号２０）。シグナル配列（１－１９）、ＮＹＡ－１１４３（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-1143 full-length amino acid sequence (SEQ ID NO: 20). Signal sequence (1-19), NYA-1143 (21-266), FLAG-His tag (267-292). ＮＹＡ－２１４３全長のアミノ酸配列（配列番号２１）。シグナル配列（１－１９）、ＮＹＡ－２１４３（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2143 full-length amino acid sequence (SEQ ID NO: 21). Signal sequence (1-19), NYA-2143 (21-266), FLAG-His tag (267-292). ＮＹＡ－２０３５全長のアミノ酸配列（配列番号２２）。シグナル配列（１－１９）、ＮＹＡ－２０３５（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2035 full-length amino acid sequence (SEQ ID NO: 22). Signal sequence (1-19), NYA-2035 (21-266), FLAG-His tag (267-292). ＮＹＡ－１１４３－ＶＬ０１のアミノ酸配列（配列番号２３）。Amino acid sequence of NYA-1143-VL01 (SEQ ID NO: 23). ＮＹＡ－２０４４全長のアミノ酸配列（配列番号２４）。シグナル配列（１－１９）、ＮＹＡ－２０４４（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2044 full-length amino acid sequence (SEQ ID NO: 24). Signal sequence (1-19), NYA-2044 (21-266), FLAG-His tag (267-292). ＮＹＡ－２０４５全長のアミノ酸配列（配列番号２５）。シグナル配列（１－１９）、ＮＹＡ－２０４５（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2045 full-length amino acid sequence (SEQ ID NO: 25). Signal sequence (1-19), NYA-2045 (21-266), FLAG-His tag (267-292). ＮＹＡ－２０４７全長のアミノ酸配列（配列番号２６）。シグナル配列（１－１９）、ＮＹＡ－２０４７（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2047 full-length amino acid sequence (SEQ ID NO: 26). Signal sequence (1-19), NYA-2047 (21-266), FLAG-His tag (267-292). ＮＹＡ－２０４８全長のアミノ酸配列（配列番号２７）。シグナル配列（１－１９）、ＮＹＡ－２０４８（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）NYA-2048 full-length amino acid sequence (SEQ ID NO: 27). Signal sequence (1-19), NYA-2048 (21-266), FLAG-His tag (267-292) ＮＹＡ－２０６０全長のアミノ酸配列（配列番号２８）。シグナル配列（１－１９）、ＮＹＡ－２０６０（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2060 full-length amino acid sequence (SEQ ID NO: 28). Signal sequence (1-19), NYA-2060 (21-266), FLAG-His tag (267-292). ＮＹＡ－２０６１全長のアミノ酸配列（配列番号２９）。シグナル配列（１－１９）、ＮＹＡ－２０６１（２１ー２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-2061 full-length amino acid sequence (SEQ ID NO: 29). Signal sequence (1-19), NYA-2061 (21-266), FLAG-His tag (267-292). ＮＹＡ－０００１全長のアミノ酸配列（配列番号３０）。シグナル配列（１－１９）、ＮＹＡ－０００１（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）NYA-0001 full-length amino acid sequence (SEQ ID NO: 30). Signal sequence (1-19), NYA-0001 (21-266), FLAG-His tag (267-292) ＨＣ１全長のアミノ酸配列（配列番号３１）。シグナル配列（１－１９）、Ｈｉｎｇｅ（２０－２９）、ＣＨ２（３０－１３９）、ＣＨ３（１４０－２４６）。Amino acid sequence of HC1 full length (SEQ ID NO: 31). Signal sequence (1-19), Hinge (20-29), CH2 (30-139), CH3 (140-246). ＮＹＦ－００１６－ＨＣ２全長のアミノ酸配列（配列番号３２）。シグナル配列（１－１９）、ＮＹＡ－１１４３（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0016-HC2 full-length amino acid sequence (SEQ ID NO: 32). Signal sequence (1-19), NYA-1143 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００１９－ＨＣ２全長のアミノ酸配列（配列番号３３）。シグナル配列（１－１９）、ＮＹＡ－２１４３（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0019-HC2 full-length amino acid sequence (SEQ ID NO: 33). Signal sequence (1-19), NYA-2143 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００２２－ＨＣ２全長のアミノ酸配列（配列番号３４）。シグナル配列（１－１９）、ＮＹＡ－１１６３（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0022-HC2 full-length amino acid sequence (SEQ ID NO: 34). Signal sequence (1-19), NYA-1163 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００２３－ＨＣ２全長のアミノ酸配列（配列番号３５）。シグナル配列（１－１９）、ＮＹＡ－２０２３（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0023-HC2 full-length amino acid sequence (SEQ ID NO: 35). Signal sequence (1-19), NYA-2023 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００２７－ＨＣ２全長のアミノ酸配列（配列番号３６）。シグナル配列（１－１９）、ＮＹＡ－２０２７（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0027-HC2 full-length amino acid sequence (SEQ ID NO: 36). Signal sequence (1-19), NYA-2027 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００３５－ＨＣ２全長のアミノ酸配列（配列番号３７）。シグナル配列（１－１９）、ＮＹＡ－２０３５（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0035-HC2 full-length amino acid sequence (SEQ ID NO: 37). Signal sequence (1-19), NYA-2035 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００４４－ＨＣ２全長のアミノ酸配列（配列番号３８）。シグナル配列（１－１９）、ＮＹＡ－２０４４（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0044-HC2 full-length amino acid sequence (SEQ ID NO: 38). Signal sequence (1-19), NYA-2044 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００４５－ＨＣ２全長のアミノ酸配列（配列番号３９）。シグナル配列（１－１９）、ＮＹＡ－２０４５（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0045-HC2 full-length amino acid sequence (SEQ ID NO: 39). Signal sequence (1-19), NYA-2045 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００４７－ＨＣ２全長のアミノ酸配列（配列番号４０）。シグナル配列（１－１９）、ＮＹＡ－２０４７（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0047-HC2 full-length amino acid sequence (SEQ ID NO: 40). Signal sequence (1-19), NYA-2047 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００４８－ＨＣ２全長のアミノ酸配列（配列番号４１）。シグナル配列（１－１９）、ＮＹＡ－２０４８（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0048-HC2 full-length amino acid sequence (SEQ ID NO: 41). Signal sequence (1-19), NYA-2048 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００６０－ＨＣ２全長のアミノ酸配列（配列番号４２）。シグナル配列（１－１９）、ＮＹＡ－２０６０（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0060-HC2 full-length amino acid sequence (SEQ ID NO: 42). Signal sequence (1-19), NYA-2060 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００６１－ＨＣ２全長のアミノ酸配列（配列番号４３）。シグナル配列（１－１９）、ＮＹＡ－２０６１（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0061-HC2 full-length amino acid sequence (SEQ ID NO: 43). Signal sequence (1-19), NYA-2061 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＡ－０００１－Ｆａｂ－ＨＣ１－κ　ｄｅｌｅｔｅ全長のアミノ酸配列（配列番号４４）。シグナル配列（１－１９），　ＮＹＡ－０００１＿ＶＨ（２０－１３９）、ＣＨ１（１４０－２３７）、Ｈｉｎｇｅ（２３８－２５２）、ＣＨ２（２５３－３６２）、ＣＨ３（３６３－４６８）。NYA-0001-Fab-HC1-κ delete full-length amino acid sequence (SEQ ID NO: 44). Signal sequence (1-19), NYA-0001_VH (20-139), CH1 (140-237), Hinge (238-252), CH2 (253-362), CH3 (363-468). ＮＹＡ－０００１－ＬＣ全長のアミノ酸配列（配列番号４５）。シグナル配列（１－２０）、ＮＹＡ－０００１＿ＶＬ（２１－１３１），ＣＬ（１３２－２３７）。NYA-0001-LC full-length amino acid sequence (SEQ ID NO: 45). Signal sequence (1-20), NYA-0001_VL (21-131), CL (132-237). Ｃ３Ｅ－７０３４のアミノ酸配列（配列番号４６）。Ｃ３Ｅ－７０３４（１－２６７）、ｓｃＦｖ（２－２４１）、ＦＬＡＧ－Ｈｉｓタグ（２４２－２６７）。Amino acid sequence of C3E-7034 (SEQ ID NO: 46). C3E-7034 (1-267), scFv (2-241), FLAG-His tag (242-267). Ｃ３Ｅ－７０３６のアミノ酸配列（配列番号４７）。Ｃ３Ｅ－７０３６（１－２６９）、ＦＬＡＧ－Ｈｉｓタグ（２４４－２６９）。Amino acid sequence of C3E-7036 (SEQ ID NO: 47). C3E-7036 (1-269), FLAG-His tag (244-269). Ｃ３Ｅ－７０８５のアミノ酸配列（配列番号４８）。Ｃ３Ｅ－７０８５（１－２６７）、ｓｃＦｖ（２－２４１）、ＦＬＡＧ－Ｈｉｓタグ（２４２－２６７）。Amino acid sequence of C3E-7085 (SEQ ID NO: 48). C3E-7085 (1-267), scFv (2-241), FLAG-His tag (242-267). Ｃ３Ｅ－７０８８のアミノ酸配列（配列番号４９）。Ｃ３Ｅ－７０８８（１－２６９）、ＦＬＡＧ－Ｈｉｓタグ（２４４－２６９）。Amino acid sequence of C3E-7088 (SEQ ID NO: 49). C3E-7088 (1-269), FLAG-His tag (244-269). Ｃ３Ｅ－７０９３のアミノ酸配列（配列番号５０）。Ｃ３Ｅ－７０９３（１－２６９）、ＦＬＡＧ－Ｈｉｓタグ（２４４－２６９）。Amino acid sequence of C3E-7093 (SEQ ID NO: 50). C3E-7093 (1-269), FLAG-His tag (244-269). Ｃ３Ｅ－７０７８のアミノ酸配列（配列番号５１）。Ｃ３Ｅ－７０７８（１－２６９）、ＦＬＡＧ－Ｈｉｓタグ（２４４－２６９）。Amino acid sequence of C3E-7078 (SEQ ID NO: 51). C3E-7078 (1-269), FLAG-His tag (244-269). ＮＹＦ－００１４－ＨＣ２全長のアミノ酸配列（配列番号５２）。シグナル配列（１－１９）、ＮＹＡ－０００１（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0014-HC2 full-length amino acid sequence (SEQ ID NO: 52). Signal sequence (1-19), NYA-0001 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００８２－ＨＣ２全長のアミノ酸配列（配列番号５３）。シグナル配列（１－１９）、ＮＹＡ－００８２（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0082-HC2 full-length amino acid sequence (SEQ ID NO: 53). Signal sequence (1-19), NYA-0082 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＦ－００８３－ＨＣ２全長のアミノ酸配列（配列番号５４）。シグナル配列（１－１９）、ＮＹＡ－２０６１（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０９５（２７２－５１１）、ｌｉｎｋｅｒ（５１２－５１３）、Ｈｉｎｇｅ（５１４－５２８）、ＣＨ２（５２９－６３８）、ＣＨ３（６３９－７４５）。NYF-0083-HC2 full-length amino acid sequence (SEQ ID NO: 54). Signal sequence (1-19), NYA-2061 (21-266), linker (267-271), C3E-7095 (272-511), linker (512-513), Hinge (514-528), CH2 (529) -638), CH3 (639-745). ＮＹＺ－００８２－ＨＣ２全長のアミノ酸配列（配列番号５５）。シグナル配列（１－１９）、ＮＹＡ－３０６１（２１－２７１）、ｌｉｎｋｅｒ（２７２－２７６）、Ｃ３Ｅ－７０９６（２７７－５１６）、ｌｉｎｋｅｒ（５１７－５１８）、Ｈｉｎｇｅ（５１９－５３３）、ＣＨ２（５３４－６４３）、ＣＨ３（６４４－７５０）。NYZ-0082-HC2 full-length amino acid sequence (SEQ ID NO: 55). Signal sequence (1-19), NYA-3061 (21-271), linker (272-276), C3E-7096 (277-516), linker (517-518), Hinge (519-533), CH2 (534) -643), CH3 (644-750). ＮＹＺ－００８３－ＨＣ２全長のアミノ酸配列（配列番号５６）。シグナル配列（１－１９）、ＮＹＡ－３０６１（２１－２７１）、ｌｉｎｋｅｒ（２７２－２７６）、Ｃ３Ｅ－７０９７（２７７－５１６）、ｌｉｎｋｅｒ（５１７－５１８）、Ｈｉｎｇｅ（５１９－５３３）、ＣＨ２（５３４－６４３）、ＣＨ３（６４４－７５０）。NYZ-0083-HC2 full-length amino acid sequence (SEQ ID NO: 56). Signal sequence (1-19), NYA-3061 (21-271), linker (272-276), C3E-7097 (277-516), linker (517-518), Hinge (519-533), CH2 (534) -643), CH3 (644-750). ＮＹＺ－１０１０－ＨＣ２全長のアミノ酸配列（配列番号５７）。シグナル配列（１－１９）、ＮＹＡ－３０６１（２１－２７１）、ｌｉｎｋｅｒ（２７２－２７６）、Ｃ３Ｅ－７０８５－ＶＨ（２７７－３９４），　ＣＨ１（３９５－４９２）Ｈｉｎｇｅ（４９３－５０７）、ＣＨ２（５０８－６１７）、ＣＨ３（６１８－７２４）。NYZ-1010-HC2 full-length amino acid sequence (SEQ ID NO: 57). Signal sequence (1-19), NYA-3061 (21-271), linker (272-276), C3E-7085-VH (277-394), CH1 (395-492) Hinge (493-507), CH2 ( 508-617), CH3 (618-724). Ｃ３Ｅ－７０８５－ＬＣ全長のアミノ配列（配列番号５８）。シグナル配列（１－２０）、Ｃ３Ｅ－７０８５＿ＶＬ（２１－１２７），ＣＬ（１２８－２３３）。C3E-7085-LC full-length amino sequence (SEQ ID NO: 58). Signal sequence (1-20), C3E-7085_VL (21-127), CL (128-233). ＮＹＡ－３０６１全長のアミノ酸配列（配列番号５９）。シグナル配列（１－１９）、ＮＹＡ－３０６１（２１ー２７１）、ＦＬＡＧ－Ｈｉｓタグ（２７２－２９７）。NYA-3061 full-length amino acid sequence (SEQ ID NO: 59). Signal sequence (1-19), NYA-3061 (21-271), FLAG-His tag (272-297). ＮＹＺ－１００７－ＨＣ２全長のアミノ酸配列（配列番号６０）。シグナル配列（１－１９）、ＮＹＡ－２０６１（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５－ＶＨ（２７２－３８９）、ＣＨ１（３９０－４８７）、Ｈｉｎｇｅ（４８８－５０２）、ＣＨ２（５０３－６１２）、ＣＨ３（６１３－７１９）。NYZ-1007-HC2 full-length amino acid sequence (SEQ ID NO: 60). Signal sequence (1-19), NYA-2061 (21-266), linker (267-271), C3E-7085-VH (272-389), CH1 (390-487), Hinge (488-502), CH2 (503-612), CH3 (613-719). ＮＹＺ－１０１７－ＨＣ２全長のアミノ酸配列（配列番号６１）。シグナル配列（１－１９）、ＮＹＡ－２０４７（２１－２６６）、ｌｉｎｋｅｒ（２６７－２７１）、Ｃ３Ｅ－７０８５－ＶＨ（２７２－３８９）　、ＣＨ１（３９０－４８７）、Ｈｉｎｇｅ（４８８－５０２）、ＣＨ２（５０３－６１２）、ＣＨ３（６１３－７１９）。NYZ-1017-HC2 full-length amino acid sequence (SEQ ID NO: 61). Signal sequence (1-19), NYA-2047 (21-266), linker (267-271), C3E-7085-VH (272-389), CH1 (390-487), Hinge (488-502), CH2 (503-612), CH3 (613-719). Ｃ３Ｅ－７０９６全長アミノ酸配列（配列番号６２）。C3E-7096 full-length amino acid sequence (SEQ ID NO: 62). Ｃ３Ｅ－７０９７全長アミノ酸配列（配列番号６３）。C3E-7097 full-length amino acid sequence (SEQ ID NO: 63). Ｃ３Ｅ－７０９８全長アミノ酸配列（配列番号６４）。C3E-7098 full-length amino acid sequence (SEQ ID NO: 64). Ｃ３Ｅ－７０９９全長アミノ酸配列（配列番号６５）。C3E-7099 full-length amino acid sequence (SEQ ID NO: 65). ＮＹ－ＥＳＯのアミノ酸配列（配列番号６６）。Amino acid sequence of NY-ESO (SEQ ID NO: 66). ＨＬＡ－Ａ＊０２０１（ＧｅｎＢａｎｋ：ＡＳＡ４７５３４．１）のトランケート体のアミノ酸配列（配列番号６７）。Amino acid sequence of the truncated form of HLA-A*0201 (GenBank: ASA47534.1) (SEQ ID NO: 67). β２－マイクログロブリンのアミノ酸配列（配列番号６８）。Amino acid sequence of β2-microglobulin (SEQ ID NO: 68). ＮＹＡ－１１４３－ＶＨ０２のアミノ酸配列（配列番号６９）。Amino acid sequence of NYA-1143-VH02 (SEQ ID NO: 69). ＮＹＡ－１１４３－ＶＨ０３のアミノ酸配列（配列番号７０）。Amino acid sequence of NYA-1143-VH03 (SEQ ID NO: 70). ＮＹＡ－１１５４全長のアミノ酸配列（配列番号７１）。シグナル配列（１－１９）、ＮＹＡ－１１５４（２１－２６６）、ＦＬＡＧ－Ｈｉｓタグ（２６７－２９２）。NYA-1154 full-length amino acid sequence (SEQ ID NO: 71). Signal sequence (1-19), NYA-1154 (21-266), FLAG-His tag (267-292). 図７２（１）は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、ｌｅｎｖａｔｉｎｉｂとＰｅｍｂｒｏｌｉｚｕｍａｂ併用投与、及びＮＹＺ－１０１０とｌｅｎｖａｔｉｎｉｂとＰｅｍｂｒｏｌｉｚｕｍａｂの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 72 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, lenvatinib and Pembrolizumab combination administered, and NYZ-1010 in a human T cell transfer model. FIG. 3 is a diagram showing the antitumor activity of combined administration of lenvatinib and Pembrolizumab. Error bars in the figure indicate standard error (n=5). 図７２（２）はヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、ｌｅｎｖａｔｉｎｉｂとＰｅｍｂｒｏｌｉｚｕｍａｂ併用投与、及びＮＹＺ－１０１０とｌｅｎｖａｔｉｎｉｂとＰｅｍｂｒｏｌｉｚｕｍａｂの併用投与でのＤａｙ２８の腫瘍体積をベースラインとした各個体のＤａｙ５２の腫瘍体積変化率を示した図である。Figure 72 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, lenvatinib and Pembrolizumab combined, and NYZ-1010 and NYZ-1010 administered in a human T cell transfer model. It is a figure showing the tumor volume change rate on Day 52 of each individual with the tumor volume on Day 28 as the baseline when lenvatinib and Pembrolizumab were administered in combination. 図７３（１）は、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、抗マウスＰＤ－１抗体単独投与、及びＮＹＺ－１０１０と抗マウスＰＤ－１抗体の併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５～１０）を示す。Figure 73 (1) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse PD-1 antibody in a BALB/C strain human CD3ε knock-in mouse model. FIG. 3 is a diagram showing the antitumor activity of single administration and combined administration of NYZ-1010 and anti-mouse PD-1 antibody. Error bars in the figure indicate standard errors (n=5 to 10). 図７３（２）は、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、抗マウスＰＤ－１抗体単独投与、及びＮＹＺ－１０１０と抗マウスＰＤ－１抗体の併用投与での各個体の推定腫瘍体積、及び完全退縮マウスとＮａiｖｅマウスにおける、ＣＴ２６　ＮＹ－ＳＣＴ再移植後の推定腫瘍体積を示した図である。Figure 73 (2) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse PD-1 antibody in a BALB/C strain human CD3ε knock-in mouse model. A diagram showing the estimated tumor volume of each individual after single administration and combined administration of NYZ-1010 and anti-mouse PD-1 antibody, and the estimated tumor volume after CT26 NY-SCT reimplantation in completely regressed mice and naive mice. It is. 図７４（１）は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｄｕｒｖａｌｕｍａｂ単独投与、及びＮＹＺ－１０１０とＤｕｒｖａｌｕｍａｂの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 74 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Durvalumab alone, and NYZ-1010 and Durvalumab in a human T cell transfer model. FIG. 3 is a diagram showing the antitumor activity in combination administration of . Error bars in the figure indicate standard error (n=5). 図７４（２）はヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｄｕｒｖａｌｕｍａｂ単独投与、及びＮＹＺ－１０１０とＤｕｒｖａｌｕｍａｂの併用投与でのＤａｙ２４の腫瘍体積をベースラインとした各個体のＤａｙ４９の腫瘍体積変化率を示した図である。Figure 74 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Durvalumab alone, and NYZ-1010 and Durvalumab in a human T cell transfer model. It is a figure showing the tumor volume change rate on Day 49 of each individual with the tumor volume on Day 24 as the baseline in combination administration. 図７５（１）は、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、抗マウスＰＤ－Ｌ１抗体単独投与、及びＮＹＺ－１０１０と抗マウスＰＤ－Ｌ１抗体の併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５～１０）を示す。Figure 75 (1) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse PD-L1 antibody in a BALB/C strain human CD3ε knock-in mouse model. FIG. 2 is a diagram showing the antitumor activity of single administration and combined administration of NYZ-1010 and anti-mouse PD-L1 antibody. Error bars in the figure indicate standard errors (n=5 to 10). 図７５（２）は、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、抗マウスＣＴＬＡ－４抗体単独投与、及びＮＹＺ－１０１０と抗マウスＣＴＬＡ－４抗体の併用投与での各個体の推定腫瘍体積、及び完全退縮マウスとＮａiｖｅマウスにおける、ＣＴ２６　Ｍｏｃｋ再移植後の推定腫瘍体積を示した図である。Figure 75 (2) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse CTLA-4 antibody in a BALB/C strain human CD3ε knock-in mouse model. It is a diagram showing the estimated tumor volume of each individual after single administration and combined administration of NYZ-1010 and anti-mouse CTLA-4 antibody, and the estimated tumor volume after CT26 Mock reimplantation in completely regressed mice and naive mice. . 図７６（１）は、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、抗マウスＣＴＬＡ－４抗体単独投与、及びＮＹＺ－１０１０と抗マウスＣＴＬＡ－４抗体の併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５～１０）を示す。Figure 76 (1) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse CTLA-4 antibody in a BALB/C strain human CD3ε knock-in mouse model. FIG. 2 is a diagram showing the antitumor activity of single administration and combined administration of NYZ-1010 and anti-mouse CTLA-4 antibody. Error bars in the figure indicate standard errors (n=5 to 10). 図７６（２）は、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、抗マウスＣＴＬＡ－４抗体単独投与、及びＮＹＺ－１０１０と抗マウスＣＴＬＡ－４抗体の併用投与での各個体の推定腫瘍体積、及び完全退縮マウスとＮａiｖｅマウスにおける、ＣＴ２６　ＮＹ－ＳＣＴ再移植後の推定腫瘍体積を示した図である。Figure 76 (2) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse CTLA-4 antibody in a BALB/C strain human CD3ε knock-in mouse model. A diagram showing the estimated tumor volume of each individual after single administration and combined administration of NYZ-1010 and anti-mouse CTLA-4 antibody, and the estimated tumor volume after CT26 NY-SCT reimplantation in completely regressed mice and naive mice. It is. 図７７（１）は、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、抗マウスＴＩＧＩＴ抗体単独投与、及びＮＹＺ－１０１０と抗マウスＴＩＧＩＴ抗体の併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５～１０）を示す。Figure 77 (1) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse TIGIT antibody administered alone in the BALB/C strain human CD3ε knock-in mouse model. , and the antitumor activity of the combined administration of NYZ-1010 and anti-mouse TIGIT antibody. Error bars in the figure indicate standard errors (n=5 to 10). 図７７（２）は、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスモデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、抗マウスＴＩＧＩＴ抗体単独投与、及びＮＹＺ－１０１０と抗マウスＴＩＧＩＴ抗体の併用投与での各個体の推定腫瘍体積、及び完全退縮マウスとＮａiｖｅマウスにおける、ＣＴ２６　ＮＹ－ＳＣＴ再移植後の推定腫瘍体積を示した図である。Figure 77 (2) shows Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, and anti-mouse TIGIT antibody administered alone in the BALB/C strain human CD3ε knock-in mouse model. , the estimated tumor volume of each individual after combined administration of NYZ-1010 and anti-mouse TIGIT antibody, and the estimated tumor volume after reimplantation of CT26 NY-SCT in completely regressed mice and naive mice. 図７８は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｃａｒｂｏｐｌａｔｉｎ単独投与、及びＮＹＺ－１０１０とＣａｒｂｏｐｌａｔｉｎの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 78 shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Carboplatin alone, and NYZ-1010 and Carboplatin combined in a human T cell transfer model. FIG. 2 is a diagram showing the antitumor activity of Error bars in the figure indicate standard error (n=5). 図７９（１）は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、ｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉy単独投与、及びＮＹＺ－１０１０とｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉyの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 79 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, sacituzumab govitecan-hziy administered alone, and NYZ- 1010 and sacituzumab govitecan-hziy when administered in combination. FIG. Error bars in the figure indicate standard error (n=5). 図７９（２）はヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、ｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉy単独投与、及びＮＹＺ－１０１０とｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉyの併用投与でのＤａｙ６の腫瘍体積をベースラインとした各個体のＤａｙ２１の腫瘍体積変化率を示した図である。Figure 79 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone administration, sacituzumab govitecan-hziy alone administration, and NYZ-1010 in the human T cell transfer model. FIG. 3 is a diagram showing the rate of change in tumor volume on Day 21 of each individual with the tumor volume on Day 6 as the baseline in the case of combined administration of govitecan-hziy and sacituzumab. 図８０（１）は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｄｏｘｏｒｕｂｉｃｉｎ単独投与、及びＮＹＺ－１０１０とＤｏｘｏｒｕｂｉｃｉｎの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 80 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Doxorubicin alone, and NYZ-1010 and Doxorubicin in a human T cell transfer model. FIG. 3 is a diagram showing the antitumor activity in combination administration of . Error bars in the figure indicate standard error (n=5). 図８０（２）はヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｄｏｘｏｒｕｂｉｃｉｎ単独投与、及びＮＹＺ－１０１０とＤｏｘｏｒｕｂｉｃｉｎの併用投与でのＤａｙ４０の腫瘍体積をベースラインとした各個体のＤａｙ５５の腫瘍体積変化率を示した図である。Figure 80 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Doxorubicin alone, and NYZ-1010 and Doxorubicin in the human T cell transfer model. It is a figure showing the tumor volume change rate on Day 55 of each individual with the tumor volume on Day 40 as the baseline in combination administration. 図８１（１）は、ヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｄｅｃｉｔａｂｉｎｅ単独投与、及びＮＹＺ－１０１０とＤｅｃｉｔａｂｉｎｅの併用投与での抗腫瘍活性を示した図である。図中のエラーバーは標準誤差（ｎ＝５）を示す。Figure 81 (1) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Decitabine alone, and NYZ-1010 and Decitabine in a human T cell transfer model. FIG. 3 is a diagram showing the antitumor activity in combination administration of . Error bars in the figure indicate standard error (n=5). 図８１（２）はヒトＴ細胞移入モデルにおける、Ｆｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子、ＮＹＺ－１０１０単独投与、Ｄｅｃｉｔａｂｉｎｅ単独投与、及びＮＹＺ－１０１０とＤｅｃｉｔａｂｉｎｅの併用投与でのＤａｙ７の腫瘍体積をベースラインとした各個体のＤａｙ３２の腫瘍体積変化率を示した図である。Figure 81 (2) shows Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Decitabine alone, and NYZ-1010 and Decitabine in a human T cell transfer model. It is a figure showing the tumor volume change rate on Day 32 of each individual with the tumor volume on Day 7 as the baseline in combination administration. ２０２２年３月１６日出願の特願２０２２－０４１２５３の配列表Sequence list of patent application 2022-041253 filed on March 16, 2022
　以下、本発明を詳細に説明する。
１．定義
　本発明において、「遺伝子」とは、蛋白質のアミノ酸をコードする塩基配列が含まれるヌクレオチド鎖又はその相補鎖を意味し、例えば、蛋白質のアミノ酸をコードする塩基配列が含まれるヌクレオチド鎖又はその相補鎖であるポリヌクレオチド、オリゴヌクレオチド、ＤＮＡ、ｍＲＮＡ、ｃＤＮＡ、ｃＲＮＡ等は「遺伝子」の意味に含まれる。かかる遺伝子は一本鎖、二本鎖又は三本鎖以上のヌクレオチドであり、ＤＮＡ鎖とＲＮＡ鎖の会合体、一本のヌクレオチド鎖上にリボヌクレオチド（ＲＮＡ）とデオキシリボヌクレオチド（ＤＮＡ）が混在するもの及びそのようなヌクレオチド鎖を含む二本鎖又は三本鎖以上のヌクレオチドも「遺伝子」の意味に含まれる。本発明において塩基配列とヌクレオチド配列は同義である。 The present invention will be explained in detail below.
1. Definition In the present invention, the term "gene" refers to a nucleotide chain containing a base sequence encoding an amino acid of a protein or its complementary chain, such as a nucleotide chain containing a base sequence encoding an amino acid of a protein or its complementary chain. Chains such as polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc. are included in the meaning of "gene". Such a gene is a single-stranded, double-stranded, or triple-stranded or more nucleotide strand, an aggregate of a DNA strand and an RNA strand, or a mixture of ribonucleotides (RNA) and deoxyribonucleotides (DNA) on a single nucleotide strand. Also included within the meaning of "gene" are genes and double-stranded, triple-stranded or more nucleotide chains containing such nucleotide chains. In the present invention, a base sequence and a nucleotide sequence have the same meaning.
　本発明において、「ポリヌクレオチド」、「ヌクレオチド鎖」、「核酸」及び「核酸分子」は同義であり、例えば、ＤＮＡ、ＲＮＡ、プローブ、オリゴヌクレオチド、プライマー等も「ポリヌクレオチド」の意味に含まれる。かかるポリヌクレオチドは一本鎖、二本鎖又は三本以上の鎖からなるポリヌクレオチドであり、ＤＮＡ鎖とＲＮＡ鎖の会合体、一本のポリヌクレオチド鎖上にリボヌクレオチド（ＲＮＡ）とデオキシリボヌクレオチド（ＤＮＡ）が混在するもの及びそのようなポリヌクレオチド鎖を含む二本鎖又は三本以上の鎖の会合体も「ポリヌクレオチド」の意味に含まれる。 In the present invention, "polynucleotide", "nucleotide chain", "nucleic acid" and "nucleic acid molecule" have the same meaning, and for example, DNA, RNA, probe, oligonucleotide, primer, etc. are also included in the meaning of "polynucleotide". . Such polynucleotides are single-stranded, double-stranded, or polynucleotides consisting of three or more strands, such as an association of a DNA strand and an RNA strand, or a single polynucleotide strand containing ribonucleotides (RNA) and deoxyribonucleotides ( Also included within the meaning of "polynucleotide" are those containing a mixture of polynucleotides (DNA) and double-stranded or three or more stranded aggregates containing such polynucleotide chains.
　本発明において、「ポリペプチド」、「ペプチド」及び「蛋白質」は同義である。 In the present invention, "polypeptide", "peptide" and "protein" have the same meaning.
　本発明において、「抗原」を「免疫原」の意味に用いることがある。 In the present invention, "antigen" may be used to mean "immunogen."
　本発明において、「細胞」には、動物個体に由来する各種細胞、継代培養細胞、初代培養細胞、細胞株、組換え細胞及び微生物等も含まれる。 In the present invention, "cells" include various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, microorganisms, and the like.
　本発明において、「抗体」は免疫グロブリンと同義である。ただし、本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体という場合の「抗体」は、定常領域と可変領域とを有する免疫グロブリンの意味で用いる。抗体は、天然のものであるか、又は、部分的若しくは完全合成により製造された免疫グロブリンであるかは特に限定されない。本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体は後述の「分子」に含まれる。 In the present invention, "antibody" is synonymous with immunoglobulin. However, when referring to the anti-HLA/NY-ESO antibody of the present invention, the term "antibody" is used to mean an immunoglobulin having a constant region and a variable region. The antibody may be a natural immunoglobulin or a partially or completely synthetically produced immunoglobulin. The anti-HLA/NY-ESO antibody of the present invention is included in the "molecule" described below.
　本発明において、「ＮＹ－ＥＳＯペプチド」とは、ＮＹ－ＥＳＯ－１とＬＡＧＥ－１の１５７番目から１６５番目の９アミノ酸からなるペプチド（ＳＬＬＭＷＩＴＱＣ：配列番号１）を意味する。 In the present invention, the "NY-ESO peptide" means a peptide (SLLMWITQC: SEQ ID NO: 1) consisting of NY-ESO-1 and 9 amino acids from the 157th to the 165th amino acids of LAGE-1.
　本発明において、「ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ」とは、ＮＹ－ＥＳＯペプチド及びＨｉｓｔｏｃｏｍｐａｔｉｂｉｌｉｔｙ　Ｌｅｕｋｏｃｙｔｅ　Ａｎｔｉｇｅｎ－Ａ２（ＨＬＡ－Ａ２）の複合体を意味し、「ＨＬＡ／ＮＹ－ＥＳＯ」とも表記される。 In the present invention, "HLA-A2/NY-ESO" means a complex of NY-ESO peptide and Histocompatibility Leukocyte Antigen-A2 (HLA-A2), and is also written as "HLA/NY-ESO".
　本発明において、「抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ抗体」とは、ＨＬＡ－Ａ２／ＮＹ－ＥＳＯに結合する抗体、言い換えればＨＬＡ－Ａ２／ＮＹ－ＥＳＯを認識する抗体を意味する。同様に、「抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖ」とは、ＨＬＡ／ＮＹ－ＥＳＯに結合する、言い換えればＨＬＡ－Ａ２／ＮＹ－ＥＳＯを認識するｓｃＦｖを意味する。「抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ抗体」及び「抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖ」は、それぞれ「抗ＨＬＡ／ＮＹ－ＥＳＯ抗体」及び「抗ＨＬＡ／ＮＹ－ＥＳＯ　ｓｃＦｖ」とも表記される。 In the present invention, "anti-HLA-A2/NY-ESO antibody" means an antibody that binds to HLA-A2/NY-ESO, in other words, an antibody that recognizes HLA-A2/NY-ESO. Similarly, "anti-HLA-A2/NY-ESO scFv" means an scFv that binds to HLA/NY-ESO, in other words, recognizes HLA-A2/NY-ESO. "Anti-HLA-A2/NY-ESO antibody" and "anti-HLA-A2/NY-ESO scFv" are also written as "anti-HLA/NY-ESO antibody" and "anti-HLA/NY-ESO scFv," respectively.
　基本的な４鎖抗体の構造は、２つの同一な軽鎖（Ｌ鎖）、及び、２つの同一な重鎖（Ｈ鎖）から構成される。軽鎖は１つの共有ジスルフィド結合により重鎖に結合する。２つの重鎖は、重鎖のアイソタイプに応じて１つ又は複数のジスルフィド結合により互いに結合している。それぞれの軽鎖、重鎖は規則的な間隔を持つ鎖内ジスルフィド結合を持つ。重鎖と軽鎖には、アミノ酸配列が非常に高い類似性を示す定常領域とアミノ酸配列の類似性が低い可変領域とが存在する。軽鎖は、定常領域（ＣＬ）が続く可変領域（ＶＬ）をアミノ末端に有する。重鎖は３つの定常領域（ＣＨ１／ＣＨ２／ＣＨ３）が続く可変領域（ＶＨ）をアミノ末端に有する。ＶＬとＶＨは対となり、ＣＬは重鎖の第一定常領域（ＣＨ１）と並んでいる。ＶＬとＶＨは対となって、単一の抗原結合部位を形成する。 The basic four-chain antibody structure is composed of two identical light chains (L chains) and two identical heavy chains (H chains). The light chain is attached to the heavy chain by one covalent disulfide bond. The two heavy chains are linked to each other by one or more disulfide bonds, depending on the heavy chain isotype. Each light and heavy chain has regularly spaced intrachain disulfide bonds. Heavy chains and light chains have a constant region with very high amino acid sequence similarity and a variable region with low amino acid sequence similarity. Light chains have a variable region (VL) at the amino terminus followed by a constant region (CL). The heavy chain has a variable region (VH) at the amino terminus followed by three constant regions (CH1/CH2/CH3). The VL and VH are paired, and the CL is aligned with the first constant region (CH1) of the heavy chain. VL and VH pair to form a single antigen binding site.
　本発明の抗体の定常領域としては、特に限定されるものではないが、ヒトの疾患を治療又は予防するための本発明の抗体としては、好適にはヒト抗体のものが使用される。ヒト抗体の重鎖定常領域としては、例えば、Ｃγ１、Ｃγ２、Ｃγ３、Ｃγ４、Ｃμ、Ｃδ、Ｃα１、Ｃα２、Ｃε等を挙げることができる。ヒト抗体の軽鎖定常領域としては、例えば、Ｃκ、Ｃλ等を挙げることができる。 Although the constant region of the antibody of the present invention is not particularly limited, human antibodies are preferably used as the antibody of the present invention for treating or preventing human diseases. Examples of the heavy chain constant region of human antibodies include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, Cε, and the like. Examples of the light chain constant region of human antibodies include Cκ, Cλ, and the like.
　Ｆａｂは、重鎖のＶＨとそれに続くＣＨ１、及び、軽鎖のＶＬとそれに続くＣＬからなる。ＶＨとＶＬは、相補性決定領域（ＣＤＲ）を含む。 Fab consists of a heavy chain VH followed by CH1, and a light chain VL followed by CL. VH and VL contain complementarity determining regions (CDRs).
　Ｆｃ（Ｆｃ領域ともいう）は、重鎖の定常領域のカルボキシル末端領域であって、ＣＨ２とＣＨ３を含み、二量体である。本発明のＦｃは、天然の配列のＦｃであっても、天然の配列に変異がなされた変異型のＦｃ（「変異型Ｆｃ」という）であってもよく、本発明の多重特異性分子及び二重特異性分子において、好適なＦｃ領域は変異型Ｆｃであり、より好適にはヘテロ２量体を形成し得る１組のＦｃである。１組のＦｃとしては、後述の、第１ポリペプチドに含まれるＦｃ（ｉ）及び第２ポリペプチドに含まれるＦｃ（ｉｉ）の組合せをあげることができるが、１組のＦｃが会合（ヘテロ２量体形成）することができれば、それらに限定されるものではない。 Fc (also referred to as Fc region) is the carboxyl terminal region of the constant region of the heavy chain, contains CH2 and CH3, and is a dimer. The Fc of the present invention may be a natural sequence Fc or a mutant Fc in which the natural sequence is mutated (referred to as "mutant Fc"), and the multispecific molecule of the present invention and In bispecific molecules, preferred Fc regions are variant Fcs, more preferably a pair of Fcs capable of forming heterodimers. One set of Fc can be a combination of Fc (i) contained in the first polypeptide and Fc (ii) contained in the second polypeptide, which will be described later. dimer formation), it is not limited thereto.
　変異型Ｆｃとしては、ＷＯ２０１３／０６３７０２に開示される、向上した安定性を有するヘテロ多量体に含まれる改変Ｆｃ領域（ヘテロ二量体Ｆｃ領域を含む）、ＷＯ１９９６／２７０１１に開示される、異種多量体に含まれる、「突起」及び「空隙」を有するＩｇＧ抗体から誘導されるイムノグロブリンのＣＨ３領域を含むＦｃ、ＷＯ２００９／０８９００４に開示される、１以上のアミノ酸残基を荷電アミノ酸に置換することで静電的に有利であるヘテロ二量体に含まれるＣＨ３ドメインを含むＦｃ、ＷＯ２０１４／１１０６０１に開示される、立体構造変異及び／又はｐＩ（等電点）変異を用いた異種二量体に含まれる異種二量体Ｆｃ領域、ＷＯ２０１０／１５１７９２に開示される、プロテインＡへの結合を無くすか又は減少させる改変を含むＣＨ３ドメインを含む、ヘテロ二量体のＦｃ等が例示されるが、これらに限定されるものではない。 Examples of mutant Fc include modified Fc regions (including heterodimeric Fc regions) contained in heteromultimers with improved stability as disclosed in WO2013/063702, and heterogeneous Fc regions as disclosed in WO1996/27011. Fc containing the CH3 region of an immunoglobulin derived from an IgG antibody having "protrusions" and "gaps" contained in the body, and replacing one or more amino acid residues with charged amino acids, as disclosed in WO2009/089004. Fc containing a CH3 domain contained in a heterodimer, which is electrostatically advantageous, in a heterodimer using conformational variation and/or pI (isoelectric point) variation, as disclosed in WO2014/110601. Examples of the heterodimeric Fc region include a heterodimeric Fc containing a CH3 domain containing a modification that eliminates or reduces binding to protein A, as disclosed in WO2010/151792. It is not limited to.
　可変領域は、超可変領域（ＨＶＲ：ｈｙｐｅｒｖａｒｉａｂｌｅ　ｒｅｇｉｏｎ）と称される極度の可変性を有する領域と、その領域により分離されたフレームワーク領域（ＦＲ：Ｆｒａｍｅｗｏｒｋ　ｒｅｇｉｏｎ）と呼ばれる比較的不変の領域からなる。天然の重鎖と軽鎖の可変領域は、３つの超可変領域により接続される４つのＦＲを含み、各鎖の超可変領域はＦＲにより他の鎖の超可変領域とともに極近傍に保持され、抗体の抗原結合部位の形成に寄与している。 A variable region consists of an extremely variable region called a hypervariable region (HVR) and a relatively constant region called a framework region (FR) separated by this region. . The variable regions of natural heavy and light chains contain four FRs connected by three hypervariable regions, with the hypervariable regions of each chain being held in close proximity with the hypervariable regions of other chains by the FRs; Contributes to the formation of the antigen-binding site of antibodies.
　抗体分子の重鎖及び軽鎖にはそれぞれ３箇所の相補性決定領域（ＣＤＲ：Ｃｏｍｐｌｅｍｅｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ）があることが知られている。相補性決定領域は、超可変領域とも呼ばれ、抗体の重鎖及び軽鎖の可変領域内にあって、一次構造の変異性が特に高い部位であり、重鎖及び軽鎖のポリペプチド鎖の一次構造上において、通常、それぞれ３ヶ所に分離している。本発明においては、抗体の相補性決定領域について、重鎖の相補性決定領域を重鎖アミノ酸配列のアミノ末端側からＣＤＲＨ１、ＣＤＲＨ２、ＣＤＲＨ３と表記し、軽鎖の相補性決定領域を軽鎖アミノ酸配列のアミノ末端側からＣＤＲＬ１、ＣＤＲＬ２、ＣＤＲＬ３と表記する。これらの部位は立体構造の上で相互に近接し、結合する抗原に対する特異性を決定している。 It is known that the heavy chain and light chain of an antibody molecule each have three complementarity determining regions (CDRs). The complementarity-determining region, also called the hypervariable region, is located within the variable regions of the heavy and light chains of antibodies, and is a region with particularly high variability in primary structure. On the primary structure, each is usually separated into three locations. In the present invention, regarding the complementarity determining region of an antibody, the complementarity determining region of the heavy chain is expressed as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the complementarity determining region of the light chain is expressed as the light chain amino acid sequence. The sequences are expressed as CDRL1, CDRL2, and CDRL3 from the amino terminal side. These sites are structurally close to each other and determine the specificity for the antigen to which they bind.
　本発明において、ＣＤＲの位置と長さは、ＩＭＧＴの定義（Ｄｅｖｅｌｏｐｍｅｎｔａｌ　ａｎｄ　Ｃｏｍｐａｒａｔｉｖｅ　Ｉｍｍｕｎｏｌｏｇｙ　２７　（２００３）　５５－７７）により決定した。 In the present invention, the positions and lengths of CDRs were determined according to the IMGT definition (Developmental and Comparative Immunology 27 (2003) 55-77).
　ＦＲは、ＣＤＲ残基以外の可変領域である。可変領域は、一般にＦＲ１、ＦＲ２、ＦＲ３、ＦＲ４の４つのＦＲを持つ。 FR is a variable region other than CDR residues. A variable region generally has four FRs: FR1, FR2, FR3, and FR4.
　重鎖並びに軽鎖に含まれるＣＤＲとＦＲは、アミノ末端からカルボキシル末端に向かって、ＦＲＨ１－ＣＤＲＨ１－ＦＲＨ２－ＣＤＲＨ２－ＦＲＨ３－ＣＤＲＨ３－ＦＲＨ４、並びに、ＦＲＬ１－ＣＤＲＬ１－ＦＲＬ２－ＣＤＲＬ２－ＦＲＬ３－ＣＤＲＬ３－ＦＲＬ４、の順にそれぞれ配置される。 The CDRs and FRs included in the heavy chain and light chain are, from the amino terminal to the carboxyl terminal, FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3- FRL4, respectively.
　ＣＤＲとＦＲの位置は、当技術分野で周知の様々な定義、例えば、ＩＭＧＴ以外にも、Ｋａｂａｔ、Ｃｈｏｔｈｉａ、ＡｂＭ、ｃｏｎｔａｃｔ等の定義により決定することもできる。 The positions of CDRs and FRs can also be determined by various definitions well known in the art, for example, in addition to IMGT, Kabat, Chothia, AbM, contact, etc. definitions.
　本発明において、「抗体の抗原結合断片」とは、重鎖可変領域及び軽鎖可変領域から構成される、抗原との結合活性を有する抗体の部分断片を意味する。「抗体の抗原結合断片」としては、例えば、Ｆａｂ、Ｆ（ａｂ’）２、ｓｃＦｖ、Ｆａｂ’、Ｆｖ、ｓｉｎｇｌｅ－ｄｏｍａｉｎ　ａｎｔｉｂｏｄｙ（ｓｄＡｂ）等の抗原結合断片を挙げることができるが、それらに限定されるものではない。かかる抗体の抗原結合断片は、抗体蛋白質の全長分子をパパイン、ペプシン等の酵素で処理することによって得られたものに加え、組換え遺伝子を用いて適当な宿主細胞において産生された組換え蛋白質であってもよい。本発明において、「抗体の結合断片」は「抗体の抗原結合断片」と同義である。 In the present invention, the term "antigen-binding fragment of an antibody" refers to a partial fragment of an antibody that is composed of a heavy chain variable region and a light chain variable region and has antigen-binding activity. Examples of "antigen-binding fragments of antibodies" include, but are not limited to, antigen-binding fragments such as Fab, F(ab') 2 , scFv, Fab', Fv, and single-domain antibody (sdAb). It is not something that will be done. Such antigen-binding fragments of antibodies include those obtained by treating the full-length antibody protein molecules with enzymes such as papain and pepsin, as well as those obtained by recombinant proteins produced in appropriate host cells using recombinant genes. There may be. In the present invention, "antibody binding fragment" is synonymous with "antibody antigen binding fragment".
　本発明において、抗体が結合する「部位」、すなわち抗体が認識する「部位」とは、抗体が結合又は認識する抗原上の部分ペプチド又は部分高次構造を意味する。本発明においては、かかる部位のことをエピトープ、抗体の結合部位とも呼ぶ。本発明において、「変異抗体」とは、元の抗体が有するアミノ酸配列においてアミノ酸が置換、欠失、付加（付加には挿入が含まれる）（以下、「変異」と総称する）してなるアミノ酸配列を有し、且つ本発明のＨＬＡ／ＮＹ－ＥＳＯに結合するポリペプチドを意味する。かかる変異抗体における変異アミノ酸の数は、１、２、３、４、５、６、７、８、９、１０、１２、１５、２０、２５、３０、４０又は５０個である。かかる変異抗体も本発明の「抗体」に包含される。 In the present invention, the "site" to which an antibody binds, that is, the "site" recognized by an antibody, refers to a partial peptide or partial conformation on an antigen that the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site. In the present invention, a "mutated antibody" refers to amino acids formed by substitution, deletion, or addition (additions include insertions) of amino acids in the amino acid sequence of the original antibody (hereinafter collectively referred to as "mutations"). It refers to a polypeptide having the sequence and binding to the HLA/NY-ESO of the present invention. The number of mutant amino acids in such a mutant antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40 or 50. Such mutant antibodies are also included in the "antibody" of the present invention.
　本発明において、「１又は数個」における「数個」とは、２～１０個を指す。 In the present invention, "several" in "one or several" refers to 2 to 10.
　本発明において、「多重特異性分子」とは、複数の互いに異なるエピトープ、及び／又は、２つ以上の分子上の互いに異なるエピトープに結合することが可能な２つ又はそれ以上の部分を含む分子であれば特に限定されず、好適には、かかる部分のうち２つ又はそれ以上がそれぞれ抗体又はその抗原結合断片である「多重特異的抗体」であるか又は「多重特異的抗体」を含む分子である。多重特異的抗体には、１種又は２種以上の重鎖可変領域（ＶＨ）及び/又は軽鎖可変領域（ＶＬ）が含まれてよく、異なる２種類以上の重鎖及び軽鎖を有する完全長抗体すなわちＩｇＧ型多重特異的抗体であってもよく、２種類以上のＶＬ及びＶＨを有する抗原結合断片からなる抗体関連分子、例えば、Ｆａｂ、Ｆａｂ’、Ｆｖ、ｓｃＦｖ、ｓｄＡｂ等を組み合せてなるか又はそれらから派生してなる分子（ｔａｎｄｅｍ　ｓｃＦｖ、ダイアボディ、一本鎖ダイアボディ、トリアボディ等）であってもよいが、それらに限定されない。多重特異的抗体には、免疫グロブリン骨格を有さない部分が含まれていてもよい。 In the present invention, a "multispecific molecule" refers to a molecule containing multiple different epitopes and/or two or more moieties capable of binding to different epitopes on two or more molecules. Preferably, the molecule is a "multispecific antibody" or a molecule containing a "multispecific antibody", in which two or more of such parts are antibodies or antigen-binding fragments thereof, respectively, without any particular limitation. It is. A multispecific antibody may include one or more heavy chain variable regions (VH) and/or light chain variable regions (VL), and may contain a complete antibody having two or more different heavy and light chains. It may be a long antibody, that is, an IgG type multispecific antibody, and is a combination of antibody-related molecules consisting of antigen-binding fragments having two or more types of VL and VH, such as Fab, Fab', Fv, scFv, sdAb, etc. or molecules derived therefrom (tandem scFv, diabody, single chain diabody, triabody, etc.), but are not limited thereto. Multispecific antibodies may include portions that do not have an immunoglobulin backbone.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又は該抗体の抗原結合断片、あるいは、本発明の分子が奏する活性・性質としては、例えば、生物学的活性、物理化学的性質（物性ともいう）等を挙げることができ、具体的には、細胞傷害活性、ＡＤＣＣ活性、抗腫瘍活性（いずれも後述）等の各種生物活性、抗原やエピトープに対する結合活性、製造や保存時における安定性、熱安定性等の物性をあげることができる。 Examples of the activities and properties exhibited by the anti-HLA/NY-ESO antibody of the present invention or the antigen-binding fragment thereof, or the molecule of the present invention include biological activity, physicochemical properties (also referred to as physical properties), etc. Specifically, various biological activities such as cytotoxic activity, ADCC activity, and antitumor activity (all described below), binding activity to antigens and epitopes, stability during manufacturing and storage, thermal stability, etc. The physical properties of can be listed.
　本発明において、「ストリンジェントな条件下でハイブリダイズする」とは、５×ＳＳＣを含む溶液中で６５℃にてハイブリダイゼーションを行い、ついで２×ＳＳＣ－０．１％ＳＤＳを含む水溶液中で６５℃にて２０分間、０．５×ＳＳＣ－０．１％ＳＤＳを含む水溶液中で６５℃にて２０分間、並びに、０．２×ＳＳＣ－０．１％ＳＤＳを含む水溶液中で６５℃にて２０分間、それぞれ洗浄する条件又はそれと同等の条件でハイブリダイズすることを意味する。ＳＳＣとは１５０ｍＭＮａＣｌ－１５ｍＭクエン酸ナトリウムの水溶液であり、ｎ×ＳＳＣはｎ倍濃度のＳＳＣを意味する。 In the present invention, "hybridizing under stringent conditions" means hybridization is performed at 65°C in a solution containing 5xSSC, and then in an aqueous solution containing 2xSSC-0.1% SDS. 20 minutes at 65°C, 20 minutes at 65°C in an aqueous solution containing 0.5×SSC-0.1% SDS, and 65°C in an aqueous solution containing 0.2×SSC-0.1% SDS. This means hybridizing for 20 minutes under washing conditions or equivalent conditions. SSC is an aqueous solution of 150mM NaCl-15mM sodium citrate, and nxSSC means n times more concentrated SSC.
　本発明において「細胞傷害」とは、何らかの形で、細胞に病理的な変化をもたらすことを指し、直接的な外傷にとどまらず、ＤＮＡの切断や塩基の二量体の形成、染色体の切断、細胞分裂装置の損傷、各種酵素活性の低下などあらゆる細胞の構造や機能上の損傷を意味する。本発明において「細胞傷害活性」とは上記細胞傷害を引き起こすことを意味する。 In the present invention, "cytotoxicity" refers to any form of pathological change in cells, and includes not only direct trauma but also DNA cleavage, base dimer formation, chromosome breakage, It refers to any structural or functional damage to cells, such as damage to the cell division apparatus or reduction in various enzyme activities. In the present invention, "cytotoxic activity" means causing the above-mentioned cytotoxicity.
　本発明において「抗体依存性細胞傷害活性」とは、「ａｎｔｉｂｏｄｙ　ｄｅｐｅｎｄｅｎｔ　ｃｅｌｌｕｌａｒ　ｃｙｔｏｔｏｘｉｃｉｔｙ（ＡＤＣＣ）活性」を指し、ＮＫ細胞が抗体を介して腫瘍細胞等の標的細胞を傷害する作用活性を意味する。 In the present invention, "antibody-dependent cytotoxic activity" refers to "antibody dependent cellular cytotoxicity (ADCC) activity", and refers to the activity of NK cells to damage target cells such as tumor cells via antibodies.
　本発明において「Ｔ細胞リダイレクションによる細胞傷害活性」とは抗腫瘍抗原抗体と抗ＨＬＡ／ＮＹ－ＥＳＯ抗体とを含む多重特異的な分子を介して上記細胞傷害を引き起こすことを意味する。すなわち抗腫瘍抗原抗体が標的腫瘍細胞と結合し、抗ＨＬＡ／ＮＹ－ＥＳＯ抗体がＴ細胞に結合することにより標的腫瘍細胞とＴ細胞の距離を近づけ、Ｔ細胞活性化を介して細胞傷害を誘導することを意味する。該分子は、医薬組成物に含めることができる。 In the present invention, "cytotoxic activity by T cell redirection" means causing the above-mentioned cytotoxicity through multispecific molecules including anti-tumor antigen antibodies and anti-HLA/NY-ESO antibodies. In other words, the anti-tumor antigen antibody binds to target tumor cells, and the anti-HLA/NY-ESO antibody binds to T cells, thereby bringing the target tumor cells and T cells closer together and inducing cytotoxicity through T cell activation. It means to do. The molecule can be included in a pharmaceutical composition.
２．抗原
２－１．ＨＬＡ／ＮＹ－ＥＳＯ抗原　
　本発明において、「ＨＬＡ／ＮＹ－ＥＳＯ」は、ＨＬＡ／ＮＹ－ＥＳＯ蛋白質と同じ意味で用いられる。 2. Antigen 2-1. HLA/NY-ESO antigen
In the present invention, "HLA/NY-ESO" is used in the same meaning as HLA/NY-ESO protein.
　ＨＬＡ／ＮＹ－ＥＳＯは、ＨＬＡ－Ａ２、β２－Ｍｉｃｒｏｇｌｏｂｕｌｉｎ、ＮＹ－ＥＳＯペプチドの三者複合体である。ＨＬＡ－Ａ２はＨＬＡのアレルの一種であり、Ｃａｕｃａｓｉａｎで最も頻度の高いアレルとして知られる。ＨＬＡは、細胞の小胞体内でβ２－ｍｉｃｒｏｇｌｏｂｕｌｉｎと自己蛋白のペプチド断片の三者複合体を形成して細胞外に提示し、Ｔ細胞のＴＣＲ（Ｔ　Ｃｅｌｌ　Ｒｅｃｅｐｔｏｒ）に認識を受ける。ＮＹ－ＥＳＯペプチド（ＳＬＬＭＷＩＴＱＣ：配列番号６６）は、ＮＹ－ＥＳＯ－１、ＬＡＧＥ－１の１５７番目から１６５番目の９アミノ酸からなるペプチドであり、ＨＬＡ－Ａ２に提示されることが報告されている。 HLA/NY-ESO is a ternary complex of HLA-A2, β2-Microglobulin, and NY-ESO peptide. HLA-A2 is a type of HLA allele, and is known as the most frequent allele in Caucasian. HLA forms a tripartite complex of β2-microglobulin and a peptide fragment of a self-protein in the endoplasmic reticulum of cells, is presented outside the cell, and is recognized by the TCR (T Cell Receptor) of T cells. NY-ESO peptide (SLLMWITQC: SEQ ID NO: 66) is a peptide consisting of nine amino acids from positions 157 to 165 of NY-ESO-1 and LAGE-1, and is reported to be presented to HLA-A2. .
２－２．ＣＤ３抗原
　本発明において、「ＣＤ３」は、ＣＤ３蛋白質と同じ意味で用いられている。ＣＤ３は、多分子Ｔ細胞レセプター複合体の一部分としてＴ細胞上に発現され、γ鎖、δ鎖、ε鎖、ζ鎖、η鎖の５種類のポリペプチド（分子量は順に２５０００－２８０００、２１０００、２００００、１６０００、２２０００）の複合体である。ＣＤ３複合体としては、γ、δ、ε、ζ、η鎖が挙げられる。これらはサブユニットとも称される。抗ＣＤ３抗体がＴ細胞に結合することにより、Ｔ細胞活性化を介して細胞傷害を誘導する。多くの抗ＣＤ３抗体は、ＣＤ３εに結合する。ヒトＣＤ３εをコードするｃＤＮＡのヌクレオチド配列は、ＮＣＢＩ／ＧｅｎＢａｎｋにアクセッション番号：ＮＭ＿０００７３３（ＮＭ＿０００７３３．３）で、ヒトＣＤ３εのアミノ酸配列はＮＣＢＩ／ＧｅｎＰｅｐｔにアクセッション番号：ＮＰ＿０００７２４（ＮＭ＿０００７２４．１）で、それぞれ登録されている。カニクイザルＣＤ３をコードするｃＤＮＡのヌクレオチド配列は、ＧｅｎＢａｎｋにアクセッション番号：ＮＭ＿００１２８３６１５．１で登録されている。ヒトＣＤ３εのアミノ酸配列は図１０に記載されている。 2-2. CD3 Antigen In the present invention, "CD3" is used synonymously with CD3 protein. CD3 is expressed on T cells as part of a multimolecular T cell receptor complex, and consists of five types of polypeptides: γ chain, δ chain, ε chain, ζ chain, and η chain (with molecular weights of 25,000-28,000, 21,000, 20,000, 16,000, 22,000). CD3 complexes include γ, δ, ε, ζ, and η chains. These are also called subunits. Anti-CD3 antibodies bind to T cells and induce cytotoxicity through T cell activation. Many anti-CD3 antibodies bind to CD3ε. The nucleotide sequence of the cDNA encoding human CD3ε is provided to NCBI/GenBank with accession number: NM_000733 (NM_000733.3), and the amino acid sequence of human CD3ε is provided to NCBI/GenPept with accession number: NP_000724 (NM_000724.1), respectively. Registered. The nucleotide sequence of cDNA encoding cynomolgus monkey CD3 has been registered in GenBank with accession number: NM_001283615.1. The amino acid sequence of human CD3ε is shown in FIG.
２－３．抗原の調製
　本発明で用いる上述の抗原蛋白質；ＨＬＡ／ＮＹ－ＥＳＯ、ＣＤ３（以下、ＨＬＡ／ＮＹ－ＥＳＯ、ＣＤ３をまとめて、該抗原蛋白質とも記す）は、動物組織（体液を含む）、該組織由来の細胞若しくは該細胞培養物からの精製及び単離、遺伝子組換え、インビトロ翻訳、化学合成等により調製することができる。該抗原蛋白質のｃＤＮＡは、例えば、該抗原蛋白質のｍＲＮＡを発現している臓器のｃＤＮＡライブラリーを鋳型として、該抗原蛋白質のｃＤＮＡを特異的に増幅するプライマーを用いてポリメラーゼ連鎖反応（以下「ＰＣＲ」という）（Ｓａｉｋｉ，Ｒ．　Ｋ．，ｅｔ　ａｌ．，Ｓｃｉｅｎｃｅ（１９８８）２３９，４８７－４９）を行う、いわゆるＰＣＲ法により取得することができる。ヒト又はラットに発現している該抗原蛋白質をコードするヌクレオチド配列と相補的なヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件でハイブリダイズし、且つ、該抗原蛋白質と同等の生物活性を有する蛋白質をコードするポリヌクレオチドも、該抗原蛋白質のｃＤＮＡに含まれる。さらに、ヒト又はラットに発現している該抗原蛋白質遺伝子座から転写されるスプライシングバリアント又はこれにストリンジェントな条件でハイブリダイズするポリヌクレオチドであって、且つ、該抗原蛋白質と同等の生物活性を有する蛋白質をコードするポリヌクレオチドも、該抗原蛋白質のｃＤＮＡに含まれる。ヒト又はラットの該抗原蛋白質のアミノ酸配列、又はこれらの配列からシグナル配列が除かれたアミノ酸配列において、１乃至数個のアミノ酸が、置換、欠失又は付加されたアミノ酸配列からなり、該抗原蛋白質と同等の生物活性を有する蛋白質をコードするヌクレオチド配列も、該抗原蛋白質遺伝子のヌクレオチド配列に含まれる。ヒト又はラットの該抗原蛋白質の遺伝子座から転写されるスプライシングバリアントにコードされるアミノ酸配列又は該アミノ酸配列において、１又は数個のアミノ酸が、置換、欠失又は付加されたアミノ酸配列からなり、且つ、該抗原蛋白質と同等の生物活性を有する蛋白質も、該抗原蛋白質に含まれる。 2-3. Preparation of Antigen The above-mentioned antigenic proteins used in the present invention; HLA/NY-ESO, CD3 (hereinafter, HLA/NY-ESO and CD3 are collectively referred to as the antigenic proteins) can be used in animal tissues (including body fluids), It can be prepared by purification and isolation from tissue-derived cells or cell cultures, genetic recombination, in vitro translation, chemical synthesis, etc. The cDNA of the antigen protein is obtained by, for example, polymerase chain reaction (hereinafter referred to as "PCR") using a cDNA library of an organ expressing the mRNA of the antigen protein as a template and primers that specifically amplify the cDNA of the antigen protein. ” (Saiki, R. K., et al., Science (1988) 239, 487-49). A protein that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the antigenic protein expressed in humans or rats, and that has biological activity equivalent to that of the antigenic protein. The encoding polynucleotide is also included in the cDNA of the antigen protein. Furthermore, a splicing variant transcribed from the gene locus of the antigenic protein expressed in humans or rats, or a polynucleotide that hybridizes to this under stringent conditions, and which has biological activity equivalent to that of the antigenic protein. A polynucleotide encoding the protein is also included in the cDNA of the antigen protein. The amino acid sequence of the human or rat antigenic protein, or the amino acid sequence from which the signal sequence is removed, consists of an amino acid sequence in which one to several amino acids are substituted, deleted, or added, and the antigenic protein is The nucleotide sequence of the antigen protein gene also includes a nucleotide sequence encoding a protein having biological activity equivalent to that of the antigen protein gene. an amino acid sequence encoded by a splicing variant transcribed from the gene locus of the human or rat antigenic protein, or an amino acid sequence in which one or several amino acids are substituted, deleted, or added, and , a protein having biological activity equivalent to that of the antigenic protein is also included in the antigenic protein.
２－４　抗原蛋白質への結合特異性
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体及びその抗原結合断片等は、ＨＬＡ／ＮＹ－ＥＳＯを認識する。すなわち、ＨＬＡ／ＮＹ－ＥＳＯ抗原に結合する。ＨＬＡ／ＮＹ－ＥＳＯは、マウス、ラット、カニクイサル等の非ヒト動物における存在は知られていない。本発明の多重特異的抗体に含まれる抗ＣＤ３抗体及びその結合断片等は、ＣＤ３抗原を認識、すなわち、結合する。かかる抗ＣＤ３抗体等は、好適には、ヒトＣＤ３、サルＣＤ３等に結合し、より好適には、ヒトＣＤ３及びカニクイザルＣＤ３に結合する。一方、かかる好適な抗ＣＤ３抗体は、ラット及び／又はマウスのＣＤ３には結合しない。 2-4 Binding specificity to antigen protein The anti-HLA/NY-ESO antibody and antigen-binding fragment thereof of the present invention recognize HLA/NY-ESO. That is, it binds to HLA/NY-ESO antigen. HLA/NY-ESO is not known to exist in non-human animals such as mice, rats, and cynomolgus monkeys. The anti-CD3 antibody, binding fragment thereof, etc. contained in the multispecific antibody of the present invention recognize, that is, bind to the CD3 antigen. Such anti-CD3 antibodies preferably bind to human CD3, monkey CD3, etc., and more preferably bind to human CD3 and cynomolgus monkey CD3. On the other hand, such preferred anti-CD3 antibodies do not bind to rat and/or mouse CD3.
　本発明において「認識」、すなわち「結合」とは、非特異的な吸着ではない結合を意味する。認識しているか否か、すなわち、結合しているか否の判定基準としては、例えば、解離定数（Ｄｉｓｓｏｃｉａｔｉｏｎ　Ｃｏｎｓｔａｎｔ：以下、「ＫＤという」）を挙げることができる。本発明の好適な抗体等のＨＬＡ／ＮＹ－ＥＳＯ又はＣＤ３に対するＫＤ値は、１×１０－５Ｍ以下、５×１０－６Ｍ以下、２×１０－６Ｍ以下、１×１０－６Ｍ以下であり、ＨＬＡ／ＮＹ－ＥＳＯに対する好適なＫＤ値は、５×１０－７Ｍ以下、２×１０－７Ｍ以下、１×１０－７Ｍ以下、５×１０－８Ｍ以下、２×１０－８Ｍ以下、１×１０－８Ｍ以下、５×１０－９Ｍ以下、又は２×１０－９Ｍ以下であり、より好適には１×１０－９Ｍ以下である。優れた抗原結合活性を有する本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ　ｓｃＦｖとして、ＮＹＡ－１１４３、ＮＹＡ－２０２３、ＮＹＡ－２１４３、ＮＹＡ－２０４４、ＮＹＡ－２０４５、ＮＹＡ－２０６０、ＮＹＡ－２０６１、ＮＹＡ－３０６１を例示することができ、ＮＹＡ－１１４３、ＮＹＡ－２０４４、ＮＹＡ－２０４５、ＮＹＡ－２１４３及びＮＹＡ－１１５４等のＨＬＡ／ＮＹ－ＥＳＯに対するＫＤ値は１×１０－９Ｍ以下である。本発明における抗原と抗体の結合は、ＳＰＲ法、ＢＬＩ法等の生体分子間相互作用解析システム、あるいはＥＬＩＳＡ法、ＲＩＡ法等により測定又は判定することができる（ＷＯ２０２１／２００８５７参照）。本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体等のうち、優れた抗原結合特異性を有するものとして、抗ＨＬＡ／ＮＹ－ＥＳＯ　ｓｃＦｖであるＮＹＡ－０００１、ＮＹＡ－１１４３、ＮＹＡ－１１６３、ＮＹＡ－２０２３、ＮＹＡ－２０２７、ＮＹＡ－２０３５、ＮＹＡ－２０４４、ＮＹＡ－２０４５、ＮＹＡ－２０４７、ＮＹＡ－２０４８、ＮＹＡ－２０６０、ＮＹＡ－２０６１、ＮＹＡ－２１４３及びＮＹＡ－３０６１を例示することができる。 In the present invention, "recognition" or "binding" means binding that is not nonspecific adsorption. As a criterion for determining whether or not it is recognized, that is, whether or not it is bound, for example, a dissociation constant (hereinafter referred to as "KD") can be cited. The KD value of the preferred antibodies of the present invention for HLA/NY-ESO or CD3 is 1×10 −5 M or less, 5×10 −6 M or less, 2×10 −6 M or less, 1×10 −6 M The preferred KD values for HLA/NY-ESO are: 5×10 −7 M or less, 2×10 −7 M or less, 1×10 −7 M or less, 5×10 −8 M or less, 2× 10 −8 M or less, 1×10 −8 M or less, 5×10 −9 M or less, or 2×10 −9 M or less, more preferably 1×10 −9 M or less. As the anti-HLA/NY-ESO scFv of the present invention having excellent antigen binding activity, NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, NYA-2061, NYA-3061 For example, the KD value for HLA/NY-ESO such as NYA-1143, NYA-2044, NYA-2045, NYA-2143 and NYA-1154 is 1×10 −9 M or less. The binding between an antigen and an antibody in the present invention can be measured or determined by a biomolecular interaction analysis system such as an SPR method or a BLI method, or an ELISA method or an RIA method (see WO2021/200857). Among the anti-HLA/NY-ESO antibodies of the present invention, anti-HLA/NY-ESO scFv such as NYA-0001, NYA-1143, NYA-1163, NYA-2023, Examples include NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2061, NYA-2143 and NYA-3061.
３．ＨＬＡ／ＮＹ－ＥＳＯに特異的に結合する抗体又はその結合断片
３－１　抗ＨＬＡ／ＮＹ－ＥＳＯ又はその結合断片
　本発明は、ＨＬＡ／ＮＹ－ＥＳＯを認識し結合する抗体又はその結合断片を提供する。前述の通り、ＨＬＡ／ＮＹ－ＥＳＯは、ＨＬＡ－Ａ２と９ｍｅｒのＮＹ－ＥＳＯペプチド（ＳＬＬＭＷＩＴＱＣ：配列番号６６）を含む複合体である。ＮＹ－ＥＳＯペプチドは、細胞内タンパク質でありＣａｎｃｅｒ－Ｔｅｓｔｉｓ抗原であるＮＹ－ＥＳＯ－１又はＬＡＧＥ－１由来のペプチドである。ＨＬＡ／ＮＹ－ＥＳＯは、がん細胞表面に提示される。本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体及び該抗体の抗原結合断片（以下、本発明の抗体等とも記す）は、モノクローナル抗体及びポリクローナル抗体のいずれであってもよい。本発明のモノクローナル抗体のアイソタイプは、特に限定されるものではなく、例えば、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４等のＩｇＧ、ＩｇＭ、ＩｇＡ１、ＩｇＡ２等のＩｇＡ、ＩｇＤ、Ｉｇ等をあげることができる。モノクローナル抗体のアイソタイプ及びサブクラスは、例えば、オクテロニー法、ＥＬＩＳＡ法、ＲＩＡ法等で決定することができる。本発明のモノクローナル抗体としては、非ヒト動物由来の抗体（非ヒト動物抗体）、ヒト抗体、キメラ化抗体（「キメラ抗体」ともいう）、ヒト化抗体などを挙げることができ、好ましくはヒト抗体を用いることができる。本発明の抗体の範囲には、抗体の変異体（後述の「変異抗体」）も含まれ、例えば、ヒト抗体の範囲には、ヒト変異抗体も含まれる。非ヒト動物抗体としては、哺乳類、鳥類等の脊椎動物に由来する抗体などを挙げることができる。哺乳類由来の抗体としては、マウス抗体、ラット抗体などのげっ歯類由来の抗体などを挙げることができる。鳥類由来の抗体としては、ニワトリ抗体等を挙げることができる。キメラ化抗体としては、非ヒト動物抗体由来の可変領域とヒト抗体（ヒト免疫グロブリン）定常領域とを結合してなる抗体などを挙げることができるが、それらに限定されるものではない。ヒト化抗体としては、非ヒト動物抗体の可変領域中のＣＤＲをヒト抗体（ヒト免疫グロブリンの可変領域）に移植したもの、ＣＤＲに加え非ヒト動物抗体のフレームワーク領域の配列も一部ヒト抗体に移植したもの、それらのいずれかの非ヒト動物抗体由来の１つ又は２つ以上のアミノ酸をヒト型のアミノ酸で置換したものなどを挙げることができるが、それらに限定されるものではない。 3. Antibody or binding fragment thereof that specifically binds to HLA/NY-ESO 3-1 Anti-HLA/NY-ESO or binding fragment thereof The present invention provides an antibody or binding fragment thereof that recognizes and binds to HLA/NY-ESO. do. As mentioned above, HLA/NY-ESO is a complex containing HLA-A2 and a 9mer NY-ESO peptide (SLLMWITQC: SEQ ID NO: 66). NY-ESO peptide is a peptide derived from NY-ESO-1 or LAGE-1, which is an intracellular protein and a Cancer-Testis antigen. HLA/NY-ESO is presented on the surface of cancer cells. The anti-HLA/NY-ESO antibody of the present invention and its antigen-binding fragment (hereinafter also referred to as the antibody of the present invention) may be either a monoclonal antibody or a polyclonal antibody. The isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG such as IgG1, IgG2, IgG3, and IgG4, IgM, IgA such as IgA1 and IgA2, IgD, and Ig. The isotype and subclass of a monoclonal antibody can be determined, for example, by the Ochtelony method, ELISA method, RIA method, etc. The monoclonal antibodies of the present invention include antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimerized antibodies (also referred to as "chimeric antibodies"), humanized antibodies, etc., and preferably human antibodies. can be used. The scope of antibodies of the present invention also includes antibody variants (“variant antibodies” described below); for example, the scope of human antibodies also includes human variant antibodies. Examples of non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds. Examples of mammalian-derived antibodies include rodent-derived antibodies such as mouse antibodies and rat antibodies. Examples of antibodies derived from birds include chicken antibodies. Examples of chimerized antibodies include, but are not limited to, antibodies formed by combining a variable region derived from a non-human animal antibody and a constant region of a human antibody (human immunoglobulin). Humanized antibodies include those in which CDRs in the variable region of a non-human animal antibody are grafted onto a human antibody (variable region of human immunoglobulin), and in addition to the CDRs, some sequences in the framework region of a non-human animal antibody are also grafted onto a human antibody. Examples include, but are not limited to, those in which one or more amino acids derived from any of these non-human animal antibodies are substituted with human amino acids.
　抗体は、公知の種々の方法で作製することができる。公知の方法として、ハイブリドーマを用いる方法や細胞免疫法等により作製し、遺伝子組換えの手法により作製することができる。また、ヒト抗体ライブラリーより選別したファージディスプレイ由来のヒト抗体を取得する方法も知られている。例えば、ヒト抗体の可変領域をｓｃＦｖとしてファージ表面に発現させて、抗原に結合するファージを選択するファージディスプレイ法を用いることができる。抗原に結合することで選択されたファージの遺伝子を解析することによって、抗原に結合するヒト抗体の可変領域をコードするＤＮＡ配列を決定することができる。抗原に結合するｓｃＦｖのＤＮＡ配列が明らかになれば、当該配列を有する発現ベクターを作製し、適当な宿主に導入して発現させることによりヒト抗体を取得することができる（ＷＯ９２／０１０４７，ＷＯ９２／２０７９１，ＷＯ９３／０６２１３，ＷＯ９３／１１２３６，ＷＯ９３／１９１７２，ＷＯ９５／０１４３８，ＷＯ９５／１５３８８、Ａｎｎｕ．Ｒｅｖ．Ｉｍｍｕｎｏｌ（１９９４）１２，４３３－４５５）。このようにして得られた高活性を有する抗体をリード抗体として用い、該リード抗体をコードする遺伝子を変異させて、より高活性の変異体（後述の「変異抗体」）を作製することもできる。 Antibodies can be produced by various known methods. As known methods, it can be produced by a method using a hybridoma, a cell immunization method, etc., and can be produced by a genetic recombination method. Additionally, a method for obtaining phage display-derived human antibodies selected from a human antibody library is also known. For example, a phage display method can be used in which the variable region of a human antibody is expressed as an scFv on the surface of a phage and phages that bind to the antigen are selected. By analyzing the genes of phage selected for binding to the antigen, it is possible to determine the DNA sequence encoding the variable region of a human antibody that binds to the antigen. Once the DNA sequence of an scFv that binds to an antigen is known, human antibodies can be obtained by constructing an expression vector having the sequence, introducing it into an appropriate host, and expressing it (WO92/01047, WO92/ 20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, Annu. Rev. Immunol (1994) 12, 433-455). It is also possible to use the antibody with high activity obtained in this way as a lead antibody and mutate the gene encoding the lead antibody to produce a variant with higher activity (“mutant antibody” described below). .
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の重鎖に含まれるＣＤＲＨ１～ＣＤＲＨ３としては、好適には、配列番号１で表されるアミノ酸配列からなるＣＤＲＨ１、配列番号２で表されるアミノ酸配列からなるＣＤＲＨ２、並びに、列番号３で表されるアミノ酸配列、若しくは、配列番号３で表されるアミノ酸配列において６番目のアミノ酸がＮ（Ａｓｎ）であるアミノ酸配列からなる重鎖ＣＤＲＨ３の組合せをあげることができる。より好適には、配列番号１３で表されるアミノ酸配列からなるＮＹＡ－０００１重鎖可変領域、配列番号１５で表されるアミノ酸配列からなるＮＹＡ－００８２重鎖可変領域、配列番号１８で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０２３重鎖可変領域、配列番号１９で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０２７重鎖可変領域、配列番号２０で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－１１４３重鎖可変領域、配列番号１７で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－１１６３重鎖可変領域、配列番号１８で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０２３重鎖可変領域、配列番号１９で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０２７重鎖可変領域、配列番号２２で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０３５重鎖可変領域、配列番号２４で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０４４重鎖可変領域、配列番号２５で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０４５重鎖可変領域、配列番号２６で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０４７重鎖可変領域、配列番号２７で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０４８重鎖可変領域、配列番号２８で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０６０重鎖可変領域、配列番号５３で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２０６１重鎖可変領域、又は、配列番号２１で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－２１４３重鎖可変領域に含まれるＣＤＲＨ１～ＣＤＲＨ３の組合せをあげることができる。さらに、配列番号５５で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－３０６１重鎖可変領域に含まれるＣＤＲＨ１～ＣＤＲＨ３の組合せ、及び、配列番号７１で表されるアミノ酸配列のアミノ酸番号２１～１４０からなるＮＹＡ－１１５４重鎖可変領域に含まれるＣＤＲＨ１～ＣＤＲＨ３の組合せをあげることができる。 CDRH1 to CDRH3 contained in the heavy chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention are preferably CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, and CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 2. and heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 3, or the amino acid sequence in which the 6th amino acid in the amino acid sequence represented by sequence number 3 is N (Asn). I can give you some combinations. More preferably, the NYA-0001 heavy chain variable region consists of the amino acid sequence represented by SEQ ID NO: 13, the NYA-0082 heavy chain variable region consists of the amino acid sequence represented by SEQ ID NO: 15, and the NYA-0082 heavy chain variable region consists of the amino acid sequence represented by SEQ ID NO: 18. NYA-2023 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 19, NYA-2027 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 20 NYA-1143 heavy chain variable region consisting of amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 17 NYA-1163 heavy chain variable region consisting of amino acids 21-140 of the amino acid sequence represented by SEQ ID NO: 18 NYA-2023 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 19, NYA-2027 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 22 NYA-2035 heavy chain variable region consisting of amino acid numbers 21-140 of the amino acid sequence, represented by SEQ ID NO: 24 NYA-2044 heavy chain variable region consisting of amino acids 21-140 of the amino acid sequence, represented by SEQ ID NO: 25 NYA-2045 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 26 NYA-2047 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 27 NYA-2048 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence, NYA-2060 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 28, represented by SEQ ID NO: 53 CDRH1 to CDRH3 contained in the NYA-2061 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence or the NYA-2143 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 21 It is possible to list a combination of Furthermore, a combination of CDRH1 to CDRH3 contained in the NYA-3061 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 55, and amino acid number 21 of the amino acid sequence represented by SEQ ID NO: 71. Examples include the combination of CDRH1 to CDRH3 contained in the NYA-1154 heavy chain variable region consisting of ~140.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の軽鎖に含まれるＣＤＲＬ１～ＣＤＲＬ３としては、好適には、配列番号４で表されるアミノ酸配列、若しくは、配列番号４で表されるアミノ酸配列において７番目のアミノ酸がＷ（Ｔｒｐ）及び／若しくは８番目のアミノ酸がＫ（Ｌｙｓ）であるアミノ酸配列からなるＣＤＲＬ１、配列番号５で表されるアミノ酸配列からなるＣＤＲＬ２、並びに、配列番号６で表されるアミノ酸配列、若しくは、配列番号６で表されるアミノ酸配列において２番目のアミノ酸がＡ（Ａｌａ）若しくはＳ（Ｓｅｒ）であるアミノ酸配列からなるＣＤＲＬ３の組合せをあげることができる。 CDRL1 to CDRL3 contained in the light chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention preferably has the amino acid sequence represented by SEQ ID NO: 4 or the amino acid sequence represented by SEQ ID NO: 4. CDRL1 consisting of an amino acid sequence in which the seventh amino acid is W (Trp) and/or the eighth amino acid is K (Lys), CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and SEQ ID NO: 6. Examples of CDRL3 combinations include the amino acid sequence represented by SEQ ID NO: 6, or the amino acid sequence in which the second amino acid is A (Ala) or S (Ser).
　より好適には、配列番号１４で表されるアミノ酸配列からなるＮＹＡ－０００１軽鎖可変領域、配列番号１６で表されるアミノ酸配列からなるＮＹＡ－００８２軽鎖可変領域、配列番号２０で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－１１４３軽鎖可変領域、配列番号２６で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－１１６３軽鎖可変領域、配列番号１８で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０２３軽鎖可変領域、配列番号１９で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０２７軽鎖可変領域、配列番号２２で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０３５軽鎖可変領域、配列番号２４で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０４４軽鎖可変領域、配列番号２５で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０４５軽鎖可変領域、配列番号２６で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０４７軽鎖可変領域、配列番号２７で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０４８軽鎖可変領域、配列番号２８で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０６０軽鎖可変領域、配列番号２９で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２０６１軽鎖可変領域、又は、配列番号２１で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－２１４３軽鎖可変領域に含まれるＣＤＲＬ１～ＣＤＲＬ３の組合せをあげることができる。　さらに、配列番号５５で表されるアミノ酸配列のアミノ酸番号１６１～２７１からなるＮＹＡ－３０６１軽鎖可変領域に含まれるＣＤＲＬ１～ＣＤＲＬ３の組合せ、及び、配列番号７１で表されるアミノ酸配列のアミノ酸番号１５６～２６６からなるＮＹＡ－１１５４軽鎖可変領域に含まれるＣＤＲＬ１～ＣＤＲＬ３の組合せをあげることができる。 More preferably, the NYA-0001 light chain variable region consists of the amino acid sequence represented by SEQ ID NO: 14, the NYA-0082 light chain variable region consists of the amino acid sequence represented by SEQ ID NO: 16, and the NYA-0082 light chain variable region consists of the amino acid sequence represented by SEQ ID NO: 20. NYA-1143 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence, represented by SEQ ID NO: 26 NYA-1163 light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence, represented by SEQ ID NO: 18 NYA-2023 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence, NYA-2027 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 19, represented by SEQ ID NO: 22 NYA-2035 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 24 NYA-2044 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 25 NYA-2045 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 26 NYA-2047 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 27 NYA-2048 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence, represented by SEQ ID NO: 28 NYA-2060 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence, represented by SEQ ID NO: 29 CDRL1 to CDRL3 contained in the NYA-2061 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence, or the NYA-2143 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 21 It is possible to list a combination of Furthermore, a combination of CDRL1 to CDRL3 contained in the NYA-3061 light chain variable region consisting of amino acid numbers 161 to 271 of the amino acid sequence represented by SEQ ID NO: 55, and amino acid number 156 of the amino acid sequence represented by SEQ ID NO: 71. Examples include the combination of CDRL1 to CDRL3 contained in the NYA-1154 light chain variable region consisting of ~266.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の重鎖に含まれるＣＤＲＨ１～ＣＤＲＨ３及び軽鎖に含まれるＣＤＲＬ１～ＣＤＲＬ３としては、好適には、配列番号１で表されるアミノ酸配列からなるＣＤＲＨ１、配列番号２で表されるアミノ酸配列からなるＣＤＲＨ２、配列番号３で表されるアミノ酸配列、若しくは、配列番号３で表されるアミノ酸配列において６番目のアミノ酸がＮ（Ａｓｎ）であるアミノ酸配列からなる重鎖ＣＤＲＨ３、配列番号４で表されるアミノ酸配列、若しくは、配列番号４で表されるアミノ酸配列において７番目のアミノ酸がＷ（Ｔｒｐ）及び／若しくは８番目のアミノ酸がＫ（Ｌｙｓ）であるアミノ酸配列からなるＣＤＲＬ１、ＤＮＮ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなるＣＤＲＬ２、並びに、配列番号６で表されるアミノ酸配列、若しくは、配列番号６で表されるアミノ酸配列において２番目のアミノ酸がＡ（Ａｌａ）若しくはＳ（Ｓｅｒ）であるアミノ酸配列からなるＣＤＲＬ３の組合せをあげることができる。より好適には、配列番号１３及び１４でそれぞれ表されるアミノ酸配列からなるＮＹＡ－０００１重鎖可変領域及び軽鎖可変領域、配列番号２０で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－１１４３重鎖可変領域及び軽鎖可変領域、配列番号１７で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－１１６３重鎖可変領域及び軽鎖可変領域、配列番号１８で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０２３重鎖可変領域及び軽鎖可変領域、配列番号１９で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０２７重鎖可変領域及び軽鎖可変領域、配列番号２２で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０３５重鎖可変領域及び軽鎖可変領域、配列番号２４で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０４４重鎖可変領域及び軽鎖可変領域、配列番号２５で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０４５重鎖可変領域及び軽鎖可変領域、配列番号２６で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０４７重鎖可変領域及び軽鎖可変領域、配列番号２７で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０４８重鎖可変領域及び軽鎖可変領域、配列番号２８で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０６０重鎖可変領域及び軽鎖可変領域、配列番号２９で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２０６１重鎖可変領域及び軽鎖可変領域、又は、配列番号２１で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－２１４３重鎖可変領域及び軽鎖可変領域に、それぞれ含まれるＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３の組合せをあげることができる。さらに、配列番号５５で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１６１～２７１からなるＮＹＡ－３０６１重鎖可変領域及び軽鎖可変領域に、それぞれ含まれるＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３の組合せ、及び、配列番号７１で表されるアミノ酸配列のアミノ酸番号２１～１４０及び１５６～２６６からなるＮＹＡ－１１５４重鎖可変領域及び軽鎖可変領域に含まれるＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３の組合せをあげることができる。 CDRH1 to CDRH3 contained in the heavy chain and CDRL1 to CDRL3 contained in the light chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention are preferably from the amino acid sequence represented by SEQ ID NO: 1. CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 2, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid whose 6th amino acid in the amino acid sequence represented by SEQ ID NO: 3 is N (Asn). Heavy chain CDRH3 consisting of the sequence, the amino acid sequence represented by SEQ ID NO: 4, or the 7th amino acid in the amino acid sequence represented by SEQ ID NO: 4 is W (Trp) and/or the 8th amino acid is K (Lys) or , a combination of CDRL3 consisting of an amino acid sequence represented by SEQ ID NO: 6 in which the second amino acid is A (Ala) or S (Ser). More preferably, NYA-0001 heavy chain variable region and light chain variable region consisting of the amino acid sequences represented by SEQ ID NO: 13 and 14, respectively, and amino acid numbers 21-140 and 156- of the amino acid sequence represented by SEQ ID NO: 20. NYA-1143 heavy chain variable region and light chain variable region consisting of 266, NYA-1163 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 17, NYA-2023 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 18, amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 19, and NYA-2027 heavy chain variable region and light chain variable region consisting of amino acid numbers 156 to 266, NYA-2035 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 22 NYA-2044 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 24, and amino acid numbers 21 to 21 of the amino acid sequence represented by SEQ ID NO: 25. NYA-2045 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 26; Chain variable region, NYA-2048 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 27, amino acid number of the amino acid sequence represented by SEQ ID NO: 28 NYA-2060 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266, NYA-2061 heavy chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 29 and light chain variable region, or CDRH1 to CDRH3 and Combinations of CDRL1 to CDRL3 can be listed. Furthermore, a combination of CDRH1 to CDRH3 and CDRL1 to CDRL3 respectively contained in the NYA-3061 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 161 to 271 of the amino acid sequence represented by SEQ ID NO: 55, And, list the combinations of CDRH1 to CDRH3 and CDRL1 to CDRL3 contained in the NYA-1154 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 71. Can be done.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の重鎖可変領域としては、上記重鎖ＣＤＲ又はそれらの組合せを含むものを、好適な例としてあげることができ、より好適には、ＮＹＡ－０００１重鎖可変領域、ＮＹＡ－００８２重鎖可変領域、ＮＹＡ－１１４３重鎖可変領域、ＮＹＡ－１１６３重鎖可変領域、ＮＹＡ－２０２３重鎖可変領域、ＮＹＡ－２０２７重鎖可変領域、ＮＹＡ－２０３５重鎖可変領域、ＮＹＡ－２０４４重鎖可変領域、ＮＹＡ－２０４５重鎖可変領域、ＮＹＡ－２０４７重鎖可変領域、ＮＹＡ－２０４８重鎖可変領域、ＮＹＡ－２０６０重鎖可変領域、ＮＹＡ－２０６１重鎖可変領域、ＮＹＡ－２１４３重鎖可変領域、ＮＹＡ－３０６１重鎖可変領域、及び、ＮＹＡ－１１５４重鎖可変領域を例示することができる。それぞれの重鎖可変領域のアミノ酸配列は上記のとおりである。 Preferable examples of the heavy chain variable region of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned heavy chain CDRs or a combination thereof, and more preferably, NYA-0001 heavy chain variable region, NYA-0082 heavy chain variable region, NYA-1143 heavy chain variable region, NYA-1163 heavy chain variable region, NYA-2023 heavy chain variable region, NYA-2027 heavy chain variable region, NYA- 2035 heavy chain variable region, NYA-2044 heavy chain variable region, NYA-2045 heavy chain variable region, NYA-2047 heavy chain variable region, NYA-2048 heavy chain variable region, NYA-2060 heavy chain variable region, NYA-2061 heavy chain variable region Examples include the chain variable region, NYA-2143 heavy chain variable region, NYA-3061 heavy chain variable region, and NYA-1154 heavy chain variable region. The amino acid sequence of each heavy chain variable region is as described above.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の軽鎖可変領域としては、上記軽鎖ＣＤＲ又はそれらの組合せを含むものを、好適な例としてあげることができ、より好適には、ＮＹＡ－０００１軽鎖可変領域、ＮＹＡ－００８２軽鎖可変領域、ＮＹＡ－１１４３軽鎖可変領域、ＮＹＡ－１１６３軽鎖可変領域、ＮＹＡ－２０２３軽鎖可変領域、ＮＹＡ－２０２７軽鎖可変領域、ＮＹＡ－２０３５軽鎖可変領域、ＮＹＡ－２０４４軽鎖可変領域、ＮＹＡ－２０４５軽鎖可変領域、ＮＹＡ－２０４７軽鎖可変領域、ＮＹＡ－２０４８軽鎖可変領域、ＮＹＡ－２０６０軽鎖可変領域、ＮＹＡ－２０６１軽鎖可変領域、ＮＹＡ－２１４３軽鎖可変領域、ＮＹＡ－３０６１軽鎖可変領域、及び、ＮＹＡ－１１５４軽鎖可変領域を例示することができる。それぞれの軽鎖可変領域のアミノ酸配列は上記のとおりである。 Preferable examples of the light chain variable region of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned light chain CDRs or a combination thereof, and more preferably, NYA-0001 light chain variable region, NYA-0082 light chain variable region, NYA-1143 light chain variable region, NYA-1163 light chain variable region, NYA-2023 light chain variable region, NYA-2027 light chain variable region, NYA- 2035 light chain variable region, NYA-2044 light chain variable region, NYA-2045 light chain variable region, NYA-2047 light chain variable region, NYA-2048 light chain variable region, NYA-2060 light chain variable region, NYA-2061 light chain variable region Examples include the chain variable region, NYA-2143 light chain variable region, NYA-3061 light chain variable region, and NYA-1154 light chain variable region. The amino acid sequence of each light chain variable region is as described above.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の重鎖可変領域及び軽鎖可変領域としては、上記重鎖及び軽鎖のＣＤＲ又はそれらの組合せを含むものを、好適な例としてあげることができ、より好適には、ＮＹＡ－０００１重鎖可変領域及び軽鎖可変領域、ＮＹＡ－００８２重鎖可変領域及び軽鎖可変領域、ＮＹＡ－１１４３重鎖可変領域及び軽鎖可変領域、ＮＹＡ－１１６３重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０２３重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０２７重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０３５重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０４４重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０４５重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０４７重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０４８重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０６０重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２０６１重鎖可変領域及び軽鎖可変領域、ＮＹＡ－２１４３重鎖可変領域及び軽鎖可変領域の組合せ、ＮＹＡ－３０６１重鎖可変領域及び軽鎖可変領域の組合せ、及び、ＮＹＡ－１１５４重鎖可変領域及び軽鎖可変領域の組合せを例示することができる。 Preferable examples of the heavy chain variable region and light chain variable region of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned heavy chain and light chain CDRs or a combination thereof. More preferably, NYA-0001 heavy chain variable region and light chain variable region, NYA-0082 heavy chain variable region and light chain variable region, NYA-1143 heavy chain variable region and light chain variable region, NYA- 1163 heavy chain variable region and light chain variable region, NYA-2023 heavy chain variable region and light chain variable region, NYA-2027 heavy chain variable region and light chain variable region, NYA-2035 heavy chain variable region and light chain variable region, NYA-2044 heavy chain variable region and light chain variable region, NYA-2045 heavy chain variable region and light chain variable region, NYA-2047 heavy chain variable region and light chain variable region, NYA-2048 heavy chain variable region and light chain variable region region, NYA-2060 heavy chain variable region and light chain variable region, NYA-2061 heavy chain variable region and light chain variable region, NYA-2143 heavy chain variable region and light chain variable region combination, NYA-3061 heavy chain variable region and light chain variable regions, and combinations of NYA-1154 heavy chain variable regions and light chain variable regions.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の重鎖としては、上記好適又はより好適な重鎖可変領域を含むものを、好適な例としてあげることができ、より好適には、ＮＹＡ－０００１重鎖、ＮＹＡ－００８２重鎖、ＮＹＡ－１１４３重鎖、ＮＹＡ－１１６３重鎖、ＮＹＡ－２０２３重鎖、ＮＹＡ－２０２７重鎖、ＮＹＡ－２０３５重鎖、ＮＹＡ－２０４４重鎖、ＮＹＡ－２０４５重鎖、ＮＹＡ－２０４７重鎖、ＮＹＡ－２０４８重鎖、ＮＹＡ－２０６０重鎖、ＮＹＡ－２０６１重鎖、ＮＹＡ－２１４３重鎖、ＮＹＡ－３０６１重鎖、及び、ＮＹＡ－１１５４重鎖を例示することができる。 Preferred examples of the heavy chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned preferred or more preferred heavy chain variable regions, and more preferably, NYA-0001 heavy chain, NYA-0082 heavy chain, NYA-1143 heavy chain, NYA-1163 heavy chain, NYA-2023 heavy chain, NYA-2027 heavy chain, NYA-2035 heavy chain, NYA-2044 heavy chain, NYA- Examples include 2045 heavy chain, NYA-2047 heavy chain, NYA-2048 heavy chain, NYA-2060 heavy chain, NYA-2061 heavy chain, NYA-2143 heavy chain, NYA-3061 heavy chain, and NYA-1154 heavy chain. be able to.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の軽鎖としては、上記好適又はより好適な軽鎖可変領域を含むものを、好適な例としてあげることができ、より好適には、ＮＹＡ－０００１軽鎖、ＮＹＡ－００８２軽鎖、ＮＹＡ－１１４３軽鎖、ＮＹＡ－１１６３軽鎖、ＮＹＡ－２０２３軽鎖、ＮＹＡ－２０２７軽鎖、ＮＹＡ－２０３５軽鎖、ＮＹＡ－２０４４軽鎖、ＮＹＡ－２０４５軽鎖、ＮＹＡ－２０４７軽鎖、ＮＹＡ－２０４８軽鎖、ＮＹＡ－２０６０軽鎖、ＮＹＡ－２０６１軽鎖、ＮＹＡ－２１４３軽鎖、ＮＹＡ－３０６１軽鎖、及び、ＮＹＡ－１１５４軽鎖を例示することができる。 Preferred examples of the light chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned preferred or more preferred light chain variable regions, and more preferably, NYA-0001 light chain, NYA-0082 light chain, NYA-1143 light chain, NYA-1163 light chain, NYA-2023 light chain, NYA-2027 light chain, NYA-2035 light chain, NYA-2044 light chain, NYA- Examples include 2045 light chain, NYA-2047 light chain, NYA-2048 light chain, NYA-2060 light chain, NYA-2061 light chain, NYA-2143 light chain, NYA-3061 light chain, and NYA-1154 light chain. be able to.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の重鎖及び軽鎖としては、上記好適又はより好適な重鎖可変領域及び軽鎖可変領域をそれぞれ含むものを、好適な例としてあげることができ、より好適には、ＮＹＡ－０００１、ＮＹＡ－１１４３、ＮＹＡ－１１６３、ＮＹＡ－２０２３、ＮＹＡ－２０２７、ＮＹＡ－２０３５、ＮＹＡ－２０４４、ＮＹＡ－２０４５、ＮＹＡ－２０４７、ＮＹＡ－２０４８、ＮＹＡ－２０６０、ＮＹＡ－２０６１、ＮＹＡ－２１４３、ＮＹＡ－３０６１、及びＮＹＡ－１１５４の重鎖及び軽鎖の組合せを例示することができる。 Preferred examples of the heavy chain and light chain of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned preferred or more preferred heavy chain variable region and light chain variable region, respectively. More preferably, NYA-0001, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA Examples include heavy and light chain combinations of -2060, NYA-2061, NYA-2143, NYA-3061, and NYA-1154.
　抗体の抗原結合断片とは、該抗体の有する機能のうち少なくとも抗原結合性を保持する断片又はその修飾物を意味する。かかる抗体の機能としては、一般的には、抗原結合活性、抗原の活性を調節する活性、抗体依存性細胞傷害活性及び補体依存性細胞傷害活性等を挙げることができる。本発明の抗体等及び本発明の抗体等を含む多重特異的な分子とした場合の機能としては、例えばＴ細胞のリダイレクション、Ｔ細胞の活性化、Ｔ細胞を活性化することによるがん細胞の細胞傷害活性を挙げることができる。 An antigen-binding fragment of an antibody means a fragment or a modified product thereof that retains at least antigen-binding properties among the functions of the antibody. Functions of such antibodies generally include antigen-binding activity, antigen activity-regulating activity, antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, and the like. The functions of the antibodies of the present invention and multispecific molecules containing the antibodies of the present invention include, for example, T cell redirection, T cell activation, and cancer cell activation by activating T cells. Cytotoxic activity can be mentioned.
　抗体の抗原結合断片としては、該抗体の有する活性のうち少なくとも抗原結合性を保持している該抗体の断片であれば特に限定されないが、例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ、重鎖及び軽鎖のＦｖを適当なリンカーで連結させた一本鎖Ｆｖ（ｓｃＦｖ）、単一ドメイン抗体（ｓｄＡｂ）、等を挙げることができるが、それらに限定されるものではない。リンカー部分を保有するｓｃＦｖのように、本発明の抗体の抗原結合断片以外の部分を含む分子も、本発明の抗体の抗原結合断片の意味に包含される。 Antigen-binding fragments of antibodies are not particularly limited as long as they retain at least antigen-binding activity of the antibody, but examples include Fab, Fab', F(ab') 2 , Examples include, but are not limited to, Fv, single chain Fv (scFv) in which heavy chain and light chain Fv are linked with a suitable linker, and single domain antibody (sdAb). Molecules containing portions other than the antigen-binding fragment of the antibody of the present invention, such as scFv carrying a linker portion, are also included within the meaning of the antigen-binding fragment of the antibody of the present invention.
　抗体蛋白質のアミノ末端及び／又はカルボキシル末端のアミノ酸が１若しくは数個又はそれ以上を欠失してなり、且つ該抗体の有する機能の少なくとも一部を保持している分子も、抗体の抗原結合断片の意味に包含される。そのような抗体の抗原結合断片の修飾体も、本発明の抗体若しくはその抗原結合断片、又はその修飾体（後述）に包含される。 A molecule in which one or more amino acids at the amino terminus and/or carboxyl terminus of an antibody protein are deleted, and which retains at least a part of the functions of the antibody, is also an antigen-binding fragment of an antibody. included in the meaning of Modified forms of such antigen-binding fragments of antibodies are also included in the antibody or antigen-binding fragment thereof of the present invention, or modified forms thereof (described below).
　本発明の抗体又はその抗原結合断片の一つの態様は、ｓｃＦｖである。ｓｃＦｖは、抗体の重鎖可変領域と軽鎖可変領域とをポリペプチドのリンカーで連結することにより得られる（Ｐｌｕｃｋｔｈｕｎ　Ａ．Ｔｈｅ　Ｐｈａｒｍａｃｏｌｏｇｙ　ｏｆ　Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ１１３，Ｒｏｓｅｎｂｕｒｇ及びＭｏｏｒｅ編，Ｓｐｒｉｎｇｅｒ　Ｖｅｒｌａｇ，Ｎｅｗ　Ｙｏｒｋ，２６９－３１５（１９９４），Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（２００５），２３，１１２６－１１３６）。また、２つのｓｃＦｖをポリペプチドリンカーで結合させて作製されるタンデムｓｃＦｖも二重特異性分子として使用することができる。さらに３つ以上のｓｃＦｖより成るトリアボディ等も多重特異性分子として使用することができる。 One embodiment of the antibody or antigen-binding fragment thereof of the present invention is an scFv. scFv is obtained by linking the heavy chain variable region and light chain variable region of an antibody with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, edited by Rosenburg and Moore, Spring ger Verlag, New York, 269- 315 (1994), Nature Biotechnology (2005), 23, 1126-1136). Additionally, tandem scFvs produced by linking two scFvs with a polypeptide linker can also be used as bispecific molecules. Furthermore, triabodies and the like composed of three or more scFv can also be used as multispecific molecules.
　ＨＬＡ／ＮＹ－ＥＳＯに特異的なｓｃＦｖ（「抗ＨＬＡ／ＮＹ－ＥＳＯ　ｓｃＦｖ」とも呼ばれる）としては、好適には上記ＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含むもの、より好適には上記重鎖可変領域及び軽鎖可変領域を含むもの、より一層好適にはＮＹＡ－０００１（配列番号３０）で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－００８２（配列番号１５で表されるアミノ酸配列及び配列番号１６で表されるアミノ酸配列を含む）、ＮＹＡ－１１４３（配列番号２０で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－１１６３（配列番号１７で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０２３（配列番号１８で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０２７（配列番号１９で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０３５（配列番号２２で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０４４（配列番号２４で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０４５（配列番号２５で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０４７（配列番号２６で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０４８（配列番号２７で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０６０（配列番号２８で表されるアミノ酸配列のアミノ酸番号２１～２６６）、ＮＹＡ－２０６１（配列番号２９で表されるアミノ酸配列のアミノ酸番号２１～２６６）、及び、ＮＹＡ－２１４３（配列番号２１で表されるアミノ酸配列のアミノ酸番号２１～２６６）をそれぞれ例示することができる。さらに、ＮＹＡ－３０６１（配列番号５５で表されるアミノ酸配列のアミノ酸番号２１～２７１）、及び、ＮＹＡ－１１５４（配列番号７１で表されるアミノ酸配列のアミノ酸番号２１～２６６）を例示することができる。 The HLA/NY-ESO-specific scFv (also called "anti-HLA/NY-ESO scFv") preferably includes the above-mentioned CDRH1 to CDRH3 and CDRL1 to CDRL3, and more preferably the above-mentioned heavy chain variable region. and light chain variable region, more preferably NYA-0001 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 30), NYA-0082 (amino acid sequence represented by SEQ ID NO: 15), including the amino acid sequence represented by SEQ ID NO: 16), NYA-1143 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 20), NYA-1163 (amino acid numbers of the amino acid sequence represented by SEQ ID NO: 17) 21-266), NYA-2023 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 18), NYA-2027 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 19), NYA-2035 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 22), NYA-2044 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 24), NYA-2045 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 25), Amino acid numbers 21-266 of the amino acid sequence), NYA-2047 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 26), NYA-2048 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 27) ), NYA-2060 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 28), NYA-2061 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 29), and NYA-2143 ( Amino acid numbers 21 to 266) of the amino acid sequence represented by SEQ ID NO: 21 can be exemplified. Furthermore, NYA-3061 (amino acid numbers 21 to 271 of the amino acid sequence represented by SEQ ID NO: 55) and NYA-1154 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 71) may be exemplified. can.
　抗ＨＬＡ／ＮＹ－ＥＳＯ　ｓｃＦｖの好適な態様にはカルボキシル末端側にＦＬＡＧ－Ｈｉｓタグが融合したもの（単に「タグ付加体」とも呼ばれる）が含まれる。好適なタグ付加体としては、ＮＹＡ－０００１タグ付加体（配列番号３０のアミノ酸番号２０～２９２）、ＮＹＡ－１１４３タグ付加体（配列番号２０のアミノ酸番号２０～２９２）、ＮＹＡ－１１６３タグ付加体（配列番号１７のアミノ酸番号２０～２９２）、ＮＹＡ－２０２３タグ付加体（配列番号１８のアミノ酸番号２０～２９２）、ＮＹＡ－２０２７タグ付加体（配列番号１９のアミノ酸番号２０～２９２）、ＮＹＡ－２０３５タグ付加体（配列番号２２のアミノ酸番号２０～２９２）、ＮＹＡ－２０４４タグ付加体（配列番号２４のアミノ酸番号２０～２９２）、ＮＹＡ－２０４５タグ付加体（配列番号２５のアミノ酸番号２０～２９２）、ＮＹＡ－２０４７タグ付加体（配列番号２６のアミノ酸番号２０～２９２）、ＮＹＡ－２０４８タグ付加体（配列番号２７のアミノ酸番号２０～２９２）、ＮＹＡ－２０６０タグ付加体（配列番号２８のアミノ酸番号２０～２９２）、ＮＹＡ－２０６１タグ付加体（配列番号２９のアミノ酸番号２０～２９２）、ＮＹＡ－２１４３タグ付加体（配列番号２１のアミノ酸番号２０～２９２）、ＮＹＡ－３０６１ＦＬＡＧ－Ｈｉｓタグ付加体（配列番号５６のアミノ酸番号２１～２７１のカルボキシル末端に配列番号２２のアミノ酸番号２６７～２９２が付加してなるアミノ酸配列）、及び、ＮＹＡ－１１５４タグ付加体（配列番号７１のアミノ酸番号２１～２９２）をあげることができる。 A preferred embodiment of the anti-HLA/NY-ESO scFv includes one in which a FLAG-His tag is fused to the carboxyl terminal side (also simply referred to as a "tag adduct"). Suitable tag adducts include NYA-0001 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 30), NYA-1143 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 20), and NYA-1163 tag adduct. (amino acid numbers 20-292 of SEQ ID NO: 17), NYA-2023 tag adduct (amino acid numbers 20-292 of SEQ ID NO: 18), NYA-2027 tag adduct (amino acid numbers 20-292 of SEQ ID NO: 19), NYA- 2035 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 22), NYA-2044 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 24), NYA-2045 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 25) ), NYA-2047 tag adduct (amino acid numbers 20-292 of SEQ ID NO: 26), NYA-2048 tag adduct (amino acid numbers 20-292 of SEQ ID NO: 27), NYA-2060 tag adduct (amino acid numbers 20-292 of SEQ ID NO: 28) Nos. 20 to 292), NYA-2061 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 29), NYA-2143 tag adduct (amino acid numbers 20 to 292 of SEQ ID NO: 21), NYA-3061FLAG-His tag adduct (amino acid sequence formed by adding amino acid numbers 267 to 292 of SEQ ID NO: 22 to the carboxyl terminal of amino acid numbers 21 to 271 of SEQ ID NO: 56), and NYA-1154 tag adduct (amino acid numbers 21 to 292 of SEQ ID NO: 71) ) can be given.
　それらのうち、ＮＹＡ－２０２３及びそのタグ付加体、ＮＹＡ－２０４７及びそのタグ付加体、ＮＹＡ－２０４８及びそのタグ付加体、ＮＹＡ－２０６０及びそのタグ付加体、並びに、ＮＹＡ－２０６１及びそのタグ付加体は、Ｆｃ付加型二重特異性分子において、優れた生物活性、物性等を有し、より好適である。 Among them, NYA-2023 and its tagged product, NYA-2047 and its tagged product, NYA-2048 and its tagged product, NYA-2060 and its tagged product, and NYA-2061 and its tagged product. is more suitable as an Fc-added bispecific molecule because it has excellent biological activity, physical properties, etc.
　また、抗ＨＬＡ／ＮＹ－ＥＳＯ　ｓｃＦｖ及びそのタグ付加体を宿主細胞において発現させる場合、そのアミノ末端にシグナルペプチドを付加させることができる。シグナルペプチドが付加した抗ＨＬＡ／ＮＹ－ＥＳＯ　ｓｃＦｖタグ付加体のアミノ酸配列としては、配列番号３０、２０、１７～１９、２２、２４、２５、２６～２９、２１、及び、配列番号５６のアミノ酸番号２１～２７１のカルボキシル末端にＦＬＡＧ－Ｈｉｓタグ（配列番号２２のアミノ酸番号２６７～２９２）が付加してなるアミノ酸配列をあげることができる。 Furthermore, when anti-HLA/NY-ESO scFv and its tagged product are expressed in host cells, a signal peptide can be added to the amino terminus. The amino acid sequences of the anti-HLA/NY-ESO scFv tag adduct with a signal peptide include the amino acids of SEQ ID NO: 30, 20, 17-19, 22, 24, 25, 26-29, 21, and SEQ ID NO: 56. An amino acid sequence in which a FLAG-His tag (amino acid numbers 267 to 292 of SEQ ID NO: 22) is added to the carboxyl terminus of numbers 21 to 271 can be mentioned.
　ｓｃＦｖは、抗体の可変領域を一本鎖抗体（ｓｃＦｖ）としてファージ表面に発現させて、抗原に結合するファージを選択するファージディスプレイ法（Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（２００５），２３，（９），ｐ．１１０５－１１１６）により取得することができる。抗原に結合することで選択されたファージの遺伝子を解析することによって、抗原に結合するヒト抗体の可変領域をコードするＤＮＡ配列を決定することができる。抗原に結合するｓｃＦｖのＤＮＡ配列が明らかになれば、当該配列を有する発現ベクターを作製し、適当な宿主に導入して発現させることによりヒト抗体を取得することができる（ＷＯ９２／０１０４７、ＷＯ９２／２０７９１、ＷＯ９３／０６２１３、ＷＯ９３／１１２３６、ＷＯ９３／１９１７２、ＷＯ９５／０１４３８、ＷＯ９５／１５３８８、Ａｎｎｕ．Ｒｅｖ．Ｉｍｍｕｎｏｌ（１９９４）１２，ｐ．４３３－４５５、Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（２００５）２３（９），ｐ．１１０５－１１１６）。 scFv is a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105) in which the variable region of an antibody is expressed on the surface of a phage as a single chain antibody (scFv) and phages that bind to the antigen are selected. -1116). By analyzing the genes of phage selected for binding to the antigen, it is possible to determine the DNA sequence encoding the variable region of a human antibody that binds to the antigen. Once the DNA sequence of an scFv that binds to an antigen is known, human antibodies can be obtained by constructing an expression vector containing the sequence, introducing it into an appropriate host, and expressing it (WO92/01047, WO92/ 20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, Annu.Rev.Immunol (1994) 12, p.433-455, Nature Biotechnology (2005) ) 23(9), p. 1105-1116).
　本発明の抗体は、単一の重鎖可変領域を有し、軽鎖配列を有さない抗体であってもよい。このような抗体は、単一ドメイン抗体（ｓｉｎｇｌｅ　ｄｏｍａｉｎ　ａｎｔｉｂｏｄｙ：ｓｄＡｂ）又はナノボディ（ｎａｎｏｂｏｄｙ）と呼ばれており、抗原結合能が保持されていることが報告されている（Ｍｕｙｌｄｅｍａｎｓ　Ｓ．ｅｔ．ａｌ．，Ｐｒｏｔｅｉｎ　Ｅｎｇ．，（１９９４）７（９），１１２９－３５，Ｈａｍｅｒｓ－Ｃａｓｔｅｒｍａｎ　Ｃ．ｅｔ．ａｌ．，Ｎａｔｕｒｅ（１９９３）３６３（６４２８），４４６－４４８）。これらの抗体も、本発明における抗体の抗原結合断片の意味に包含される。 The antibody of the present invention may have a single heavy chain variable region and no light chain sequence. Such antibodies are called single domain antibodies (sdAbs) or nanobodies, and it has been reported that they retain antigen-binding ability (Muyldemans S. et. al. , Protein Eng., (1994) 7(9), 1129-35, Hamers-Casterman C. et. al., Nature (1993) 363(6428), 446-448). These antibodies are also included within the meaning of antigen-binding fragments of antibodies in the present invention.
　また、本発明には、抗体の重鎖及び軽鎖の全長配列を適切なリンカーを用いて連結した一本鎖イムノグロブリン（ｓｉｎｇｌｅ　ｃｈａｉｎ　ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ）が含まれる（Ｌｅｅ，Ｈ－Ｓ，ｅｔ．ａｌ．，Ｍｏｌｅｃｕｌａｒ　Ｉｍｍｕｎｏｌｏｇｙ（１９９９）３６，６１－７１；Ｓｈｉｒｒｍａｎｎ，Ｔ．ｅｔ．ａｌ．，ｍＡｂｓ（２０１０），２（１），１－４）。このような一本鎖イムノグロブリンは二量体化することによって、本来は四量体である抗体と類似した構造と活性を保持することが可能である。本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体は、一本鎖イムノグロブリンであってもよい。 The present invention also includes a single chain immunoglobulin in which the full-length sequences of the heavy chain and light chain of an antibody are linked using an appropriate linker (Lee, HS, et. al. , Molecular Immunology (1999) 36, 61-71; Shirrmann, T. et. al., mAbs (2010), 2(1), 1-4). By dimerizing, such single-chain immunoglobulins can maintain a structure and activity similar to those of antibodies, which are originally tetramers. The anti-HLA/NY-ESO antibody of the invention may be a single chain immunoglobulin.
　本発明のｓｃＦｖにおいて、重鎖可変領域および軽鎖可変領域がジスルフィド結合していてもよい。 In the scFv of the present invention, the heavy chain variable region and the light chain variable region may be disulfide bonded.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体は、ＨＬＡ／ＮＹ－ＥＳＯに結合すれば、複数の異なる抗体に由来する部分から構成される抗体であってもよく、例えば、複数の異なる抗体間で重鎖及び／又は軽鎖を交換したもの、重鎖及び／又は軽鎖の全長を交換したもの、可変領域のみ又は定常領域のみを交換したもの、ＣＤＲの全部又は一部のみを交換したもの等を挙げることができる。キメラ化抗体の重鎖可変領域と軽鎖可変領域は、異なる本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体に由来してもよい。ヒト化抗体の重鎖及び軽鎖の可変領域中の重鎖ＣＤＲ１乃至重鎖ＣＤＲ３並びに軽鎖ＣＤＲ１乃至軽鎖ＣＤＲ３は、２種又はそれ以上の本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体に由来してもよい。ヒト抗体の重鎖及び軽鎖の可変領域中の重鎖ＣＤＲ１乃至重鎖ＣＤＲ３並びに軽鎖ＣＤＲ１乃至軽鎖ＣＤＲ３は、２種又はそれ以上の本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体が有するＣＤＲの組合せであってもよい。 The anti-HLA/NY-ESO antibody of the present invention may be an antibody composed of parts derived from a plurality of different antibodies, as long as it binds to HLA/NY-ESO. Those in which the chain and/or light chain are exchanged, those in which the entire length of the heavy chain and/or light chain are exchanged, those in which only the variable region or only the constant region are exchanged, those in which only all or part of the CDRs are exchanged, etc. can be mentioned. The heavy chain variable region and light chain variable region of the chimerized antibody may be derived from different anti-HLA/NY-ESO antibodies of the invention. Heavy chain CDR1 to heavy chain CDR3 and light chain CDR1 to light chain CDR3 in the heavy chain and light chain variable regions of the humanized antibody are derived from two or more anti-HLA/NY-ESO antibodies of the present invention. It's okay. Heavy chain CDR1 to heavy chain CDR3 and light chain CDR1 to light chain CDR3 in the heavy chain and light chain variable regions of human antibodies are the CDRs possessed by two or more anti-HLA/NY-ESO antibodies of the present invention. It may be a combination.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体には、本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体に含まれるアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチドの相補鎖とストリンジェントな条件下でハイブリダイズするポリヌクレオチドに含まれるヌクレオチド配列によりコードされるアミノ酸配列を含み、且つ、ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体も含まれる。 The anti-HLA/NY-ESO antibody of the present invention is hybridized under stringent conditions with a complementary strand of a polynucleotide containing a nucleotide sequence encoding the amino acid sequence contained in the anti-HLA/NY-ESO antibody of the present invention. Also included are antibodies that contain the amino acid sequence encoded by the nucleotide sequence contained in the polynucleotide and that bind to HLA/NY-ESO.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片の重鎖可変領域に含まれるアミノ酸配列（好適には、配列番号１８で表されるアミノ酸配列のアミノ酸番号２１～１４０のアミノ酸配列、配列番号６９又は配列番号７０で表されるアミノ酸配列、配列番号３９で表されるアミノ酸配列、又は、配列番号５７、配列番号６０もしくは配列番号６１で表されるアミノ酸配列のアミノ酸番号２１～１４０のアミノ酸配列）、及び／又は、軽鎖可変領域に含まれるアミノ酸配列（好適には、配列番号１８で表されるアミノ酸配列のアミノ酸番号１５６～２６６のアミノ酸配列、配列番号２８で表されるアミノ酸配列のアミノ酸番号１５６～２６６のアミノ酸配列、配列番号２３で表されるアミノ酸配列、配列番号５７で表されるアミノ酸配列のアミノ酸番号１６１～２７１のアミノ酸配列、又は、配列番号６０もしくは配列番号６１で表されるアミノ酸配列のアミノ酸番号１５６～２６６のアミノ酸配列）と、９０％、９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％、又は９９％以上同一のアミノ酸配列を含み、且つＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片であってもよい。 The amino acid sequence contained in the heavy chain variable region of the anti-HLA/NY-ESO antibody or antigen-binding fragment thereof of the present invention (preferably the amino acid sequence of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 18, The amino acid sequence represented by No. 69 or SEQ ID No. 70, the amino acid sequence represented by SEQ ID No. 39, or the amino acid number 21 to 140 of the amino acid sequence represented by SEQ ID No. 57, SEQ ID No. 60, or SEQ ID No. 61. sequence), and/or the amino acid sequence contained in the light chain variable region (preferably the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 18, the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28) The amino acid sequence of amino acid numbers 156 to 266, the amino acid sequence represented by SEQ ID NO: 23, the amino acid sequence of amino acids 161 to 271 of the amino acid sequence represented by SEQ ID NO: 57, or the amino acid sequence represented by SEQ ID NO: 60 or SEQ ID NO: 61. Amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to and an antibody or an antigen-binding fragment thereof that binds to HLA/NY-ESO.
　軽鎖可変領域の位置と長さは、ＩＭＧＴの定義により決定する場合と比べ、ＩＭＧＴとは異なる定義（例えば、Ｋａｂａｔ、Ｃｈｏｔｈｉａ、ＡｂＭ，ｃｏｎｔａｃｔ等）を用いて決定した場合、ＩＭＧＴの定義により決定した該軽鎖可変領域アミノ酸配列のカルボキシル末端に、さらに、１つ又は２つ以上のアミノ酸、例えば、アルギニンやグリシンが含まれることがある。このような軽鎖可変領域を有する抗体又はその結合断片も本発明の抗体又はその結合断片に包含される。 The position and length of the light chain variable region is determined by the IMGT definition when it is determined using a definition different from IMGT (e.g. Kabat, Chothia, AbM, contact, etc.) than when it is determined by the IMGT definition. The carboxyl terminus of the light chain variable region amino acid sequence may further include one or more amino acids, such as arginine or glycine. Antibodies or binding fragments thereof having such light chain variable regions are also included in the antibodies or binding fragments thereof of the present invention.
　本発明の抗体等としては、本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体の結合断片に変異を導入して、ＨＬＡ／ＮＹ－ＥＳＯ、特にヒト及び／又はカニクイザルのＨＬＡ／ＮＹ－ＥＳＯに対する結合能を最適化させたものであってもよい。変異を導入する具体的な方法として、エラープローンＰＣＲ法を用いたランダム突然変異誘発法、ＮＮＫライブラリーを用いた部位特異的アミノ酸変異導入、構造情報を利用した部位特異的変異導入、及びそれら組合せを挙げることができる。 The antibodies and the like of the present invention have the ability to bind to HLA/NY-ESO, particularly human and/or cynomolgus monkey HLA/NY-ESO, by introducing mutations into the binding fragment of the anti-HLA/NY-ESO antibody of the present invention. It may be an optimized one. Specific methods for introducing mutations include random mutagenesis using error-prone PCR, site-specific amino acid mutagenesis using NNK libraries, site-specific mutagenesis using structural information, and combinations thereof. can be mentioned.
３－２．抗ＨＬＡ／ＮＹ－ＥＳＯ抗体の変異体（変異抗体）
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体の変異抗体には、好適には、蛋白質の分解若しくは酸化に対する感受性の低下、生物活性や機能の維持、改善若しくは低下や変化の抑制、抗原結合能の改善若しくは調節、又は物理化学的性質若しくは機能的性質の付与等がなされ得る。蛋白質は、その表面にある特定のアミノ酸側鎖が変化して当該蛋白質の機能や活性が変化することが知られ、そのような例には、アスパラギン側鎖の脱アミド化、アスパラギン酸側鎖の異性化等が含まれる。そのようなアミノ酸側鎖の変化を防ぐために別のアミノ酸に置き換えたものも、本発明の変異抗体の範囲に含まれる。 3-2. Variant of anti-HLA/NY-ESO antibody (mutant antibody)
The mutant antibody of the anti-HLA/NY-ESO antibody of the present invention preferably has a reduced sensitivity to protein degradation or oxidation, maintains, improves, or suppresses a decline or change in biological activity or function, and improves antigen binding ability. or adjustment, or imparting physicochemical properties or functional properties, etc. It is known that changes in specific amino acid side chains on the surface of proteins can change the function and activity of the protein. Examples of such changes include deamidation of asparagine side chains and deamidation of aspartate side chains. Includes isomerization, etc. Mutant antibodies of the present invention also include antibodies in which other amino acids are substituted to prevent such changes in amino acid side chains.
　本発明の変異抗体の例として、抗体の有するアミノ酸配列において保存的アミノ酸置換されてなるアミノ酸配列を有する抗体を挙げることができる。保存的アミノ酸置換とは、アミノ酸側鎖に関連のあるアミノ酸グループ内で生じる置換である。 An example of the mutant antibody of the present invention includes an antibody having an amino acid sequence resulting from conservative amino acid substitutions in the amino acid sequence of the antibody. Conservative amino acid substitutions are those that occur within a group of amino acids that are related in their amino acid side chains.
　好適なアミノ酸グループは、以下の通りである：酸性グループ＝アスパラギン酸、グルタミン酸；塩基性グループ＝リジン、アルギニン、ヒスチジン；非極性グループ＝アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン；及び非帯電極性ファミリー＝グリシン、アスパラギン、グルタミン、システイン、セリン、スレオニン、チロシン。他の好適なアミノ酸グループは次のとおりである：脂肪族ヒドロキシグループ＝セリン及びスレオニン；アミド含有グループ＝アスパラギン及びグルタミン；脂肪族グループ＝アラニン、バリン、ロイシン及びイソロイシン；並びに芳香族グループ＝フェニルアラニン、トリプトファン及びチロシン。かかる変異抗体におけるアミノ酸置換は、元の抗体が有する抗原結合活性を低下させない範囲で行うのが好ましい。 Suitable amino acid groups are: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar families = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other suitable amino acid groups are: aliphatic hydroxy groups = serine and threonine; amide-containing groups = asparagine and glutamine; aliphatic groups = alanine, valine, leucine and isoleucine; and aromatic groups = phenylalanine, tryptophan. and tyrosine. Amino acid substitutions in such mutant antibodies are preferably carried out within a range that does not reduce the antigen-binding activity of the original antibody.
　ＮＹＡ－２０２３等の本発明の抗体が有するアミノ酸配列において保存的アミノ酸置換及び／又はその他の変異がなされたアミノ酸配列を有し、且つ、ＨＬＡ／ＮＹ－ＥＳＯに結合する変異抗体、それらの抗原結合断片、それらを含む分子等；ＮＹＡ－２０２３等を含む本発明の抗体由来のＣＤＲＨ１乃至ＣＤＲＨ３及びＣＤＲＬ１乃至ＣＤＲＬ３のいずれかのアミノ酸配列において保存的アミノ酸置換及び／又はその他の変異がなされたアミノ酸配列を有し、且つ、ＨＬＡ／ＮＹ－ＥＳＯに結合する該ＣＤＲを含むキメラ化抗体、ヒト化抗体、ヒト抗体、それらの抗原結合断片、それらを含む分子等も本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体、その抗原結合断片、その変異体（変異抗体又はその抗原結合断片）、又は本発明の分子に包含される。 Mutant antibodies that have an amino acid sequence in which conservative amino acid substitutions and/or other mutations have been made in the amino acid sequence of the antibody of the present invention such as NYA-2023, and that bind to HLA/NY-ESO, and their antigen binding Fragments, molecules containing them, etc.; Chimerized antibodies, humanized antibodies, human antibodies, antigen-binding fragments thereof, molecules containing them, etc., which contain the CDRs and which bind to HLA/NY-ESO, are also included in the anti-HLA/NY-ESO antibodies of the present invention. , an antigen-binding fragment thereof, a variant thereof (a mutant antibody or an antigen-binding fragment thereof), or a molecule of the present invention.
３－３．抗ＨＬＡ／ＮＹ－ＥＳＯ抗体の結合断片
　本発明の一つの態様として、本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体の抗原結合断片（以下、単に「結合断片」という）を提供する。本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体の結合断片には、キメラ化抗体、ヒト化抗体、又は、ヒト抗体の結合断片が包含される。抗体の結合断片とは、該抗体の有する機能のうち少なくとも抗原結合性を保持する断片又はその修飾物を意味する。かかる抗体の機能としては、一般的には、抗原結合活性、抗原の活性を調節する活性（例えばアゴニスト活性）、抗原を細胞内に内在化させる活性、抗原と相互作用する物質との相互作用を阻害若しくは促進する活性等を挙げることができる。 3-3. Anti-HLA/NY-ESO Antibody Binding Fragment One aspect of the present invention provides an antigen-binding fragment (hereinafter simply referred to as "binding fragment") of the anti-HLA/NY-ESO antibody of the present invention. The binding fragments of anti-HLA/NY-ESO antibodies of the present invention include binding fragments of chimerized antibodies, humanized antibodies, or human antibodies. The antibody binding fragment means a fragment or a modified version thereof that retains at least antigen-binding properties among the functions of the antibody. The functions of such antibodies generally include antigen-binding activity, activity to modulate antigen activity (for example, agonist activity), activity to internalize the antigen into cells, and interaction with substances that interact with the antigen. Examples include activities that inhibit or promote.
　抗体の結合断片としては、該抗体の有する活性のうち少なくとも抗原結合性を保持している該抗体の断片であれば特に限定されない。このような抗体の結合断片として、例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２Ｆａｂ軽鎖のカルボキシル末端とＦａｂ重鎖のアミノ末端とが適当なリンカーで連結された一本鎖Ｆａｂ（ｓｃＦａｂ）、Ｆｖ、重鎖及び軽鎖のＦｖが適当なリンカーで連結された一本鎖Ｆｖ（ｓｃＦｖ）、単一の重鎖可変領域を有し軽鎖配列を有さない単一ドメイン抗体（ｓｄＡｂ）、又はナノボディ（ｎａｎｏｂｏｄｙ）とも呼ばれる。（Ｍｕｙｌｄｅｍａｎｓ　Ｓ．ｅｔ．ａｌ．，Ｐｒｏｔｅｉｎ　Ｅｎｇ．，（１９９４）７（９），１１２９－３５，Ｈａｍｅｒｓ－Ｃａｓｔｅｒｍａｎ　Ｃ．ｅｔ．ａｌ．，Ｎａｔｕｒｅ（１９９３）３６３（６４２８），４４６－４４８）］等を挙げることができるが、それらに限定されるものではない。リンカー部分を保有するｓｃＦａｂ、ｓｃＦｖのように、本発明の抗体の結合断片以外の部分を含む分子も、本発明の抗体の結合断片の意味に包含される。 The antibody binding fragment is not particularly limited as long as it is a fragment of the antibody that retains at least the antigen binding activity of the antibody. Such antibody binding fragments include, for example, Fab, Fab', F(ab') 2 single chain Fab (scFab) in which the carboxyl terminus of Fab light chain and the amino terminus of Fab heavy chain are linked with an appropriate linker. ), Fv, single chain Fv (scFv) in which heavy and light chain Fv are linked with a suitable linker, single domain antibody (sdAb) with a single heavy chain variable region and no light chain sequence. ) or also called nanobody. (Muyldemans S.et.al., Protein Eng., (1994) 7(9), 1129-35, Hamers-Casterman C.et.al., Nature (1993) 363(6428), 446-448)], etc. Examples include, but are not limited to. Molecules containing portions other than the antibody-binding fragment of the present invention, such as scFab and scFv having a linker portion, are also included within the meaning of the antibody-binding fragment of the present invention.
３－４．抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその結合断片の修飾体、複合体
　本発明は、抗体又はその結合断片の修飾体を提供する。本発明の抗体又はその結合断片の修飾体とは、本発明の抗体又はその結合断片に化学的又は生物学的な修飾が施されてなるものを意味する。化学的な修飾体には、アミノ酸骨格への化学部分の結合、Ｎ－結合又はＯ－結合炭水化物鎖の化学修飾体等が含まれる。生物学的な修飾体には、翻訳後修飾（例えば、Ｎ－結合又はＯ－結合への糖鎖付加、アミノ末端領域又はカルボキシル末端領域のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化）されたもの、原核生物宿主細胞を用いて発現させることによりアミノ末端にメチオニン残基が付加したもの等が含まれる。また、本発明の抗体又は抗原の検出又は単離を可能にするために標識されたもの、例えば、酵素標識体、蛍光標識体、アフィニティー標識体もかかる修飾物の意味に含まれる。このような本発明の抗体又はその結合断片の修飾物は、元の本発明の抗体又はその結合断片の安定性及び血中滞留性の改善、抗原性の低減、かかる抗体又は抗原の検出又は単離等に有用である。 3-4. Modified products and complexes of anti-HLA/NY-ESO antibodies or binding fragments thereof The present invention provides modified products of antibodies or binding fragments thereof. A modified antibody of the present invention or a binding fragment thereof means an antibody of the present invention or a binding fragment thereof that has been chemically or biologically modified. Chemical modifications include attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like. Biological modifications include post-translational modifications such as N- or O-linked glycosylation, processing of amino- or carboxyl-terminal regions, deamidation, aspartic acid isomerization, methionine oxidized), and those with a methionine residue added to the amino terminus by expression using prokaryotic host cells. Furthermore, those labeled to enable detection or isolation of the antibody or antigen of the present invention, such as enzyme labels, fluorescent labels, and affinity labels, are also included in the meaning of such modifications. Such modified antibodies of the present invention or binding fragments thereof can be used to improve the stability and retention in blood of the original antibodies of the present invention or binding fragments thereof, to reduce antigenicity, to detect such antibodies or antigens, or to Useful for separation.
　化学的修飾体に含まれる化学部分としては、ポリエチレングリコール（ＰＥＧ）、エチレングリコール／プロピレングリコールコポリマー、カルボキシメチルセルロース、デキストラン、ポリビニルアルコール等の水溶性ポリマーを例示することができる。 Examples of the chemical moiety included in the chemically modified product include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, and polyvinyl alcohol.
　生物学的な修飾物としては、酵素処理や細胞処理等により修飾が施されたもの、遺伝子組換えによりタグ等他のペプチドが付加された融合体、並びに内因性又は外来性の糖鎖修飾酵素を発現する細胞を宿主として調製されたもの等を挙げることができる。 Biologically modified products include those modified by enzyme treatment or cell treatment, fusions with other peptides such as tags added through genetic recombination, and endogenous or exogenous glycosylation enzymes. Examples include those prepared using cells expressing the expression as a host.
　かかる修飾は、抗体又はその結合断片における任意の位置に、又は所望の位置において施されてもよく、１つ又は２つ以上の位置に同一又は２種以上の異なる修飾がなされてもよい。 Such modifications may be made at any position or at a desired position in the antibody or binding fragment thereof, and the same or two or more different modifications may be made at one or more positions.
　しかし、これらの重鎖配列の欠失、あるいは、重鎖又は軽鎖配列の修飾は、抗体の抗原結合能及びエフェクター機能（補体の活性化や抗体依存性細胞傷害作用など）にはあまり影響を及ぼさない。 However, deletion of these heavy chain sequences or modifications of heavy or light chain sequences have little effect on the antigen-binding ability and effector functions of antibodies (such as complement activation and antibody-dependent cytotoxicity). does not affect
　従って、本発明には当該欠失又は修飾を受けた抗体も含まれる。例えば、重鎖カルボキシル末端において１又は２つのアミノ酸が欠失した欠失体（Ｊｏｕｒｎａｌ　ｏｆ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ　Ａ；７０５；１２９－１３４（１９９５））、重鎖カルボキシル末端のグリシン、リジンの２アミノ酸残基が欠失し、新たにカルボキシル末端に位置するプロリン残基がアミド化された当該欠失体（Ａｎａｌｙｔｉｃａｌ　Ｂｉｏｃｈｅｍｉｓｔｒｙ，３６０：７５－８３（２００７））、抗体の重鎖又は軽鎖のアミノ末端のグルタミン又はグルタミン酸残基がピログルタミル化修飾された抗体（国際特許公開ＷＯ２０１３／１４７１５３号）等を挙げることができる（それらをまとめて「欠失体」と呼ぶ）。ただし、抗原結合能及びエフェクター機能が保たれている限り、本発明に係る抗体の重鎖及び軽鎖のカルボキシル末端の欠失体は上記の種類に限定されない。本発明に係る抗体が２本以上の鎖（例えば重鎖）を含む場合、当該２本以上の鎖（例えば重鎖）は、完全長及び上記の欠失体からなる群から選択される重鎖のいずれか一種であっても良いし、いずれか二種を組合せたものであっても良い。各欠失体の量比又は分子数比は本発明に係る抗体を産生する哺乳類培養細胞の種類及び培養条件に影響を受け得るが、本発明に係る抗体の主成分としては２本の重鎖の双方でカルボキシル末端の１つのアミノ酸残基が欠失している場合を挙げることができる。 Therefore, the present invention also includes antibodies with such deletions or modifications. For example, a deletion product in which one or two amino acids are deleted at the carboxyl terminal of the heavy chain (Journal of Chromatography A; 705; 129-134 (1995)), a deletion product in which two amino acid residues, glycine and lysine, are deleted at the carboxyl terminal of the heavy chain. glutamine or glutamic acid at the amino terminal of the heavy chain or light chain of the antibody. Antibodies whose residues have been modified by pyroglutamylation (International Patent Publication No. WO2013/147153) can be mentioned (these are collectively referred to as "deletion bodies"). However, the carboxyl terminal deletions of the heavy chain and light chain of the antibody according to the present invention are not limited to the above types, as long as the antigen binding ability and effector function are maintained. When the antibody according to the present invention comprises two or more chains (e.g. heavy chains), the two or more chains (e.g. heavy chains) are heavy chains selected from the group consisting of full-length and deletion forms as described above. It may be one of these or a combination of two of them. Although the amount ratio or molecular number ratio of each deletion product may be influenced by the type and culture conditions of cultured mammalian cells producing the antibody according to the present invention, the main components of the antibody according to the present invention are two heavy chains. One example is a case where one amino acid residue at the carboxyl terminus is deleted in both.
　さらに、本発明の抗体又はその抗原結合断片（本発明の多重特異的抗体に含まれるもの等）に、発現ベクター及び／又はシグナル配列等に由来する１乃至数個のアミノ酸がアミノ末端及び／又はカルボキシル末端に付加され（且つその一部又は全部が前記のように修飾され）ていても、所望の抗原結合活性が維持されていれば、本発明の抗体の修飾体又はその抗原結合断片の修飾体の範囲に包含され、そのような抗体又は抗原結合断片の修飾体を含む多重特異的抗体も本発明の範囲に包含される。 Furthermore, the antibody of the present invention or its antigen-binding fragment (such as those included in the multispecific antibody of the present invention) has one to several amino acids derived from the expression vector and/or signal sequence, etc. at the amino terminal and/or Modifications of the modified antibody of the present invention or antigen-binding fragment thereof, as long as the desired antigen-binding activity is maintained even if the modified antibody is added to the carboxyl terminus (and a part or all of it is modified as described above). Also included within the scope of the invention are multispecific antibodies that are within the scope of the human body and include modifications of such antibodies or antigen-binding fragments.
　本発明において「抗体又はその結合断片」は「抗体又はその抗原結合断片の修飾体」もその意味に含むものである。また、本発明の多重特異的抗体に含まれる「抗体又はその抗原結合断片」はかかる「抗体又はその抗原結合断片の修飾体」もその意味に含むものである。 In the present invention, "antibody or binding fragment thereof" also includes "modified antibody or antigen-binding fragment thereof". Furthermore, the "antibody or antigen-binding fragment thereof" included in the multispecific antibody of the present invention includes within its meaning such "modified form of the antibody or antigen-binding fragment thereof."
　また、本発明の抗体に結合している糖鎖修飾を調節すること（グリコシル化、脱フコース化等）によって、抗体依存性細胞傷害活性を増強することが可能である。抗体の糖鎖修飾の調節技術としては、国際特許公開ＷＯ９９／５４３４２号、ＷＯ００／６１７３９号、ＷＯ０２／３１１４０号等が知られているが、これらに限定されるものではない。 Furthermore, by regulating the sugar chain modification (glycosylation, defucoselation, etc.) bound to the antibody of the present invention, it is possible to enhance the antibody-dependent cytotoxic activity. As techniques for regulating sugar chain modification of antibodies, international patent publications WO99/54342, WO00/61739, WO02/31140, etc. are known, but the invention is not limited to these.
　本発明には、上述の抗体と他の分子がリンカーでつながれた複合体（Ｉｍｍｕｎｏｃｏｎｊｕｇａｔｅ）も含まれる。該抗体が放射性物質や薬理作用を有する化合物（薬物）と結合している抗体－薬物複合体の例としては、ＡＤＣ（Ａｎｔｉｂｏｄｙ－Ｄｒｕｇ　Ｃｏｎｊｕｇａｔｅ）を挙げることができる（（Ｍｅｔｈｏｄｓ　Ｍｏｌ　Ｂｉｏｌ．（２０１３）１０４５：１－２７；Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（２００５）２３，ｐ．１１３７－１１４６）。 The present invention also includes a complex (Immunoconjugate) in which the above-mentioned antibody and other molecules are connected with a linker. An example of an antibody-drug conjugate in which the antibody is bound to a radioactive substance or a compound (drug) having pharmacological action is ADC (Antibody-Drug Conjugate) ((Methods Mol Biol. (2013)). 1045:1-27; Nature Biotechnology (2005) 23, p. 1137-1146).
　更に本発明には、これらの抗体と他の機能性ポリペプチドを繋げた複合体も含まれる。このような抗体－ペプチド複合体の例としては、該抗体がアルブミン結合ポリペプチドと複合体を挙げることができる（Ｐｒｏｔｅｉｎ　Ｅｎｇ　Ｄｅｓ　Ｓｅｌ．（２０１２）（２）：８１－８）。 Furthermore, the present invention also includes complexes in which these antibodies are linked to other functional polypeptides. An example of such an antibody-peptide complex is a complex in which the antibody is combined with an albumin-binding polypeptide (Protein Eng Des Sel. (2012) (2): 81-8).
　上述の抗体の修飾体、糖鎖修飾を調節された抗体、複合体は、本発明の抗体に包含され、上述の抗体の修飾体、糖鎖修飾を調節された抗体、複合体の結合断片は、本発明の抗体の結合断片に包含される。
４．抗体の産生方法
　本発明の抗体は、例えば、重鎖可変領域をコードするＤＮＡ又は軽鎖可変領域をコードするＤＮＡを発現ベクターに挿入し、発現用の宿主細胞を該ベクターで形質転換し、宿主細胞を培養することにより、リコンビナント抗体として細胞に産生させることができる。 The above-mentioned modified antibodies, antibodies with regulated glycosylation, and complexes are included in the antibody of the present invention, and binding fragments of the above-mentioned modified antibodies, antibodies with regulated glycosylation, and conjugates are included in the antibody of the present invention. , are included in the binding fragments of the antibodies of the invention.
4. Method for producing antibodies The antibody of the present invention can be produced by, for example, inserting a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region into an expression vector, transforming a host cell for expression with the vector, and By culturing cells, recombinant antibodies can be produced in the cells.
　抗体をコードするＤＮＡは、重鎖可変領域をコードするＤＮＡと重鎖定常領域をコードするＤＮＡを連結することにより重鎖をコードするＤＮＡが得られ、さらに軽鎖可変領域をコードするＤＮＡと軽鎖定常領域をコードするＤＮＡを連結することにより軽鎖をコードするＤＮＡが得られる。 The DNA encoding the antibody is obtained by linking the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region, and the DNA encoding the heavy chain is obtained by linking the DNA encoding the heavy chain variable region and the DNA encoding the light chain constant region. DNA encoding the light chain is obtained by ligating the DNA encoding the chain constant region.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体は、上記の重鎖をコードするＤＮＡ及び軽鎖をコードするＤＮＡを発現ベクターに挿入し、宿主細胞を該ベクターで形質転換し、該宿主細胞を培養して産生させることができる。この際、上記の重鎖をコードするＤＮＡ及び軽鎖をコードするＤＮＡを同じ発現ベクターに導入し、該ベクターを用いて宿主細胞を形質転換してもよいし、重鎖をコードするＤＮＡと軽鎖をコードするＤＮＡを別々のベクターに挿入し、２つのベクターを用いて宿主細胞を形質転換してもよい。この際、重鎖定常領域をコードするＤＮＡ及び軽鎖定常領域をコードするＤＮＡを予め導入したベクターに重鎖可変領域及び軽鎖可変領域をコードするＤＮＡを導入してもよい。また、該ベクターは宿主細胞からの抗体の分泌を促進するシグナルペプチドをコードするＤＮＡを含んでいてもよい、この場合、シグナルペプチドをコードするＤＮＡと抗体をコードするＤＮＡをインフレームで連結しておく。抗体が産生された後にシグナルペプチドを除去することにより、抗体を成熟タンパク質として得ることができる。 The anti-HLA/NY-ESO antibody of the present invention can be obtained by inserting the above-mentioned heavy chain-encoding DNA and light chain-encoding DNA into an expression vector, transforming host cells with the vector, and culturing the host cells. It can be produced by At this time, the DNA encoding the heavy chain and the DNA encoding the light chain described above may be introduced into the same expression vector, and the vector may be used to transform host cells, or the DNA encoding the heavy chain and the DNA encoding the light chain may be introduced into the same expression vector. The DNA encoding the strands may be inserted into separate vectors and the two vectors used to transform host cells. At this time, the DNA encoding the heavy chain variable region and the light chain variable region may be introduced into a vector into which DNA encoding the heavy chain constant region and DNA encoding the light chain constant region have been introduced in advance. Further, the vector may contain a DNA encoding a signal peptide that promotes secretion of the antibody from the host cell. In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are linked in frame. put. By removing the signal peptide after the antibody has been produced, the antibody can be obtained as a mature protein.
　この際、重鎖可変領域をコードするＤＮＡ、軽鎖可変領域をコードするＤＮＡ、重鎖可変領域をコードするＤＮＡ及び重鎖定常領域をコードするＤＮＡを連結したＤＮＡ、軽鎖可変領域をコードするＤＮＡと軽鎖定常領域をコードするＤＮＡを連結したＤＮＡをプロモータ、エンハンサー、ポリアデニル化シグナル等のエレメントと機能的に連結してもよい。ここで機能的に連結とは、エレメントがその機能を果たすように連結することをいう。 At this time, DNA encoding the heavy chain variable region, DNA encoding the light chain variable region, DNA encoding the heavy chain variable region and DNA encoding the heavy chain constant region, and DNA encoding the light chain variable region. A DNA obtained by linking a DNA and a DNA encoding a light chain constant region may be functionally linked to elements such as a promoter, an enhancer, and a polyadenylation signal. Functionally connected here means that elements are connected so that they perform their functions.
　発現ベクターは、動物細胞、細菌、酵母等の宿主中で複製可能なものであれば特に限定されず、例えば、公知のプラスミド、ファージ等が挙げられる。発現ベクターの構築に用いられるベクターとしては、例えば、ｐｃＤＮＡ（商標）（ＴｈｅｒｍｏＦｉｓｓｈｅｒ　ＳＣＩＥＮＴＩＦＩＣ社）、Ｆｌｅｘｉ（登録商標）ベクター（プロメガ社）、ｐＵＣ１９、ｐＵＥＸ２（アマシャム社製）、ｐＧＥＸ－４Ｔ、ｐＫＫ２３３－２（ファルマシア社製）、ｐＭＡＭ－ｎｅｏ（クロンテック社製）等が挙げられる。宿主細胞としては、大腸菌、枯草菌等の原核細胞も酵母、動物細胞等の真核細胞も用いることができるが、真核細胞を用いることが好ましい。例えば、動物細胞として、ヒト胎児腎細胞株であるＨＥＫ２９３細胞、チャイニーズ・ハムスター・卵巣（ＣＨＯ）細胞等を用いればよい。発現ベクターは公知の方法で宿主細胞に導入し、宿主細胞を形質転換すればよい。例えば、エレクトロポレーション法、リン酸カルシウム沈殿法、ＤＥＡＥ－デキストラントランスフェクション法等が挙げられる。産生された抗体は、通常のタンパク質で使用されている分離、精製方法を使用して精製することができる。例えば、アフィニティー・クロマトグラフィー、その他のクロマトグラフィー、フィルター、限外濾過、塩析、透析等を適宜選択し、組合せればよい。 The expression vector is not particularly limited as long as it can be replicated in a host such as animal cells, bacteria, yeast, etc., and includes, for example, known plasmids, phages, and the like. Vectors used for constructing expression vectors include, for example, pcDNA (trademark) (ThermoFissher SCIENTIFIC), Flexi (registered trademark) vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2. (manufactured by Pharmacia), pMAM-neo (manufactured by Clontech), and the like. As host cells, prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast and animal cells can be used, but eukaryotic cells are preferably used. For example, as animal cells, HEK293 cells, which are human embryonic kidney cell lines, Chinese hamster ovary (CHO) cells, etc. may be used. The expression vector may be introduced into a host cell by a known method and the host cell may be transformed. Examples include electroporation method, calcium phosphate precipitation method, DEAE-dextran transfection method, and the like. The produced antibodies can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatography, filters, ultrafiltration, salting out, dialysis, etc. may be appropriately selected and combined.
５．ＨＬＡ／ＮＹ－ＥＳＯに結合する多重特異的抗体
　本発明の多重特異的抗体は、本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片を含む。本発明の多重特異的抗体は、好適には、２以上の抗原結合部位を持つ多重特異的抗体である。すなわち、１つの分子上の２つ以上の互いに異なるエピトープ又は２つ以上の分子上の互いに異なるエピトープに結合することが可能な多重特異的抗体であり、複数の互いに異なる抗原結合断片を包む。このような多重特異的抗体には、ＩｇＧ型多重特異的抗体、２種類以上の可変領域を有する多重特異的抗体、例えばタンデムｓｃＦｖ（ｔａＦｖ）、一本鎖ダイアボディ、ダイアボディ及びトリアボディのような抗体断片、共有結合又は非共有結合して連結されている抗体断片を含むが、これらに限定されない。多重特異的抗体はＦｃを含んでいてもよい。 5. Multispecific antibodies that bind to HLA/NY-ESO Multispecific antibodies of the invention include anti-HLA/NY-ESO antibodies of the invention or antigen-binding fragments thereof. The multispecific antibody of the present invention preferably has two or more antigen binding sites. That is, it is a multispecific antibody capable of binding to two or more different epitopes on one molecule or to different epitopes on two or more molecules, and encompasses a plurality of different antigen-binding fragments. Such multispecific antibodies include IgG type multispecific antibodies, multispecific antibodies having two or more types of variable regions, such as tandem scFv (taFv), single chain diabodies, diabodies and triabodies. including, but not limited to, antibody fragments that are covalently or non-covalently linked. Multispecific antibodies may include Fc.
　本発明の多重特異的抗体は、本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその抗原結合断片に加え、１つ又は２つ以上のさらなる抗体又は該抗体の抗原結合断片を含んでよい。さらなる抗体の抗原結合断片としては、例えば、Ｆａｂ、Ｆ（ａｂ）’、Ｆｖ、ｓｃＦｖ、ｓｄＡｂを挙げることができる。 The multispecific antibody of the present invention may contain, in addition to the anti-HLA/NY-ESO antibody of the present invention or an antigen-binding fragment thereof, one or more additional antibodies or antigen-binding fragments of the antibody. Additional antigen-binding fragments of antibodies can include, for example, Fab, F(ab)', Fv, scFv, sdAb.
　本発明の好適な多重特異的抗体は、さらに、抗ＣＤ３抗体又はその抗原結合断片を含み、ＣＤ３にも特異的に結合する。 A preferred multispecific antibody of the present invention further comprises an anti-CD3 antibody or antigen-binding fragment thereof, and also specifically binds to CD3.
　本発明の多重特異的抗体に含まれる抗ＣＤ３抗体又はその抗原結合断片は、ヒトＣＤ３に結合する抗体又はその結合断片であれば特に限定されるものではないが、好適にはカニクイザル等の非ヒト霊長類のＣＤ３にも結合する。より好適な抗ＣＤ３抗体又はその抗原結合断片としては、配列番号７で表されるアミノ酸配列からなる重鎖可変領域ＣＤＲＨ１、配列番号８で表されるアミノ酸配列からなる重鎖可変領域ＣＤＲＨ２、配列番号９で表されるアミノ酸配列からなる重鎖可変領域ＣＤＲＨ３、配列番号１０で表されるアミノ酸配列からなる軽鎖可変領域ＣＤＲＬ１、ＲＤＤ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖可変領域ＣＤＲＬ２、及び、配列番号１２で表されるアミノ酸配列からなる軽鎖可変領域ＣＤＲＬ３を含んでなる抗体又はその抗原結合断片を例示することができる。 The anti-CD3 antibody or antigen-binding fragment thereof contained in the multispecific antibody of the present invention is not particularly limited as long as it is an antibody or a binding fragment thereof that binds to human CD3, but is preferably a non-human antibody such as a cynomolgus monkey. It also binds to primate CD3. More preferred anti-CD3 antibodies or antigen-binding fragments thereof include heavy chain variable region CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, heavy chain variable region CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8, and heavy chain variable region CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8. Heavy chain variable region CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9; light chain variable region CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10; ) and an antibody or an antigen-binding fragment thereof comprising a light chain variable region CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
　該ＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含むより好適な抗体又はその抗原結合断片としては、配列番号４６で表されるアミノ酸配列のアミノ酸番号２～１１９のアミノ酸配列からなるＣ３Ｅ－７０３４重鎖可変領域、配列番号４７で表されるアミノ酸配列のアミノ酸番号２～１１９のアミノ酸配列からなるＣ３Ｅ－７０３６重鎖可変領域、配列番号４８で表されるアミノ酸配列のアミノ酸番号２～１１９のアミノ酸配列からなるＣ３Ｅ－７０８５重鎖可変領域、配列番号４９で表されるアミノ酸配列のアミノ酸番号２～１１９のアミノ酸配列からなるＣ３Ｅ－７０８８重鎖可変領域、配列番号１４０で表されるアミノ酸配列のアミノ酸番号２～１１９のアミノ酸配列からなるＣ３Ｅ－７０９３重鎖可変領域、配列番号５４で表されるアミノ酸配列のアミノ酸番号２７２～３８９のアミノ酸配列からなるＣ３Ｅ－７０９６重鎖可変領域、配列番号５５で表されるアミノ酸配列のアミノ酸番号２７７～３９４のアミノ酸配列からなるＣ３Ｅ－７０９６重鎖可変領域、配列番号５６で表されるアミノ酸配列のアミノ酸番号２７７～３９４のアミノ酸配列からなるＣ３Ｅ－７０９７重鎖可変領域を含んでなる抗体又はその抗原結合断片を例示することができる。 A more preferable antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 is a C3E-7034 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 46; C3E-7036 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 47, and C3E- consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 48. 7085 heavy chain variable region, consisting of the amino acid sequence of amino acids 2 to 119 of the amino acid sequence represented by SEQ ID NO: 49, C3E-7088 heavy chain variable region consisting of the amino acid sequence of amino acids 2 to 119 of the amino acid sequence represented by SEQ ID NO: 140. C3E-7093 heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 54, C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acids 272 to 389 of the amino acid sequence represented by SEQ ID NO: 54, and the C3E-7096 heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 55. An antibody comprising a C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 277 to 394, and a C3E-7097 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 277 to 394 of the amino acid sequence represented by SEQ ID NO: 56. or an antigen-binding fragment thereof.
　また、該ＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含むより好適な抗体又はその抗原結合断片としては、配列番号４６で表されるアミノ酸配列のアミノ酸番号１３５～２４３のアミノ酸配列からなるＣ３Ｅ－７０３４軽鎖可変領域、配列番号４７で表されるアミノ酸配列のアミノ酸番号１３５～２４１のアミノ酸配列からなるＣ３Ｅ－７０３６軽鎖可変領域、配列番号４８で表されるアミノ酸配列のアミノ酸番号１３５～２４１のアミノ酸配列からなるＣ３Ｅ－７０８５軽鎖可変領域、配列番号４９で表されるアミノ酸配列のアミノ酸番号１３５～２４３のアミノ酸配列からなるＣ３Ｅ－７０８８軽鎖可変領域、配列番号５０で表されるアミノ酸配列のアミノ酸番号１３５～２４３のアミノ酸配列からなるＣ３Ｅ－７０９３軽鎖可変領域、配列番号５４で表されるアミノ酸配列のアミノ酸番号４０５～５１１のアミノ酸配列からなるＣ３Ｅ－７０９６軽鎖可変領、配列番号５５で表されるアミノ酸配列のアミノ酸番号４１０～５１６のアミノ酸配列からなるＣ３Ｅ－７０９６軽鎖可変領、配列番号５６で表されるアミノ酸配列のアミノ酸番号４１０～５１６のアミノ酸配列からなるＣ３Ｅ－７０９７軽鎖可変領を含んでなる抗体又はその抗原結合断片を例示することができる。 In addition, a more preferred antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 is C3E-7034 light chain variable consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO: 46. C3E-7036 light chain variable region, consisting of the amino acid sequence of amino acids 135 to 241 of the amino acid sequence represented by SEQ ID NO: 47, consisting of the amino acid sequence of amino acids 135 to 241 of the amino acid sequence represented by SEQ ID NO: 48 C3E-7085 light chain variable region, consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO: 49, C3E-7088 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO: 50 C3E-7093 light chain variable region consisting of the amino acid sequence of 243, C3E-7096 light chain variable region consisting of the amino acid sequence of amino acids 405 to 511 of the amino acid sequence represented by SEQ ID NO: 54, amino acid represented by SEQ ID NO: 55. A C3E-7096 light chain variable region consisting of the amino acid sequence of amino acid numbers 410 to 516 of the sequence, and a C3E-7097 light chain variable region consisting of the amino acid sequence of amino acid numbers 410 to 516 of the amino acid sequence represented by SEQ ID NO: 56. Examples include antibodies or antigen-binding fragments thereof.
　また、該ＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含むより好適な抗体又はその抗原結合断片としては、配列番号４６で表されるアミノ酸番号２～１１９及び１３５～２４３からなるＣ３Ｅ－７０３４重鎖可変領域及び軽鎖可変領域の組合せ、配列番号４７で表されるアミノ酸番号２～１１９及び１３５～２４１からなるＣ３Ｅ－７０３６重鎖可変領域及び軽鎖可変領域の組合せ、配列番号５１で表されるアミノ酸番号２～１１９及び１３５～２４３からなるＣ３Ｅ－７０７８重鎖可変領域及び軽鎖可変領域の組合せ、配列番号４８で表されるアミノ酸番号２～１１９及び１３５～２４１からなるＣ３Ｅ－７０８５重鎖可変領域及び軽鎖可変領域の組合せ、配列番号４９で表されるアミノ酸番号２～１１９及び１３５～２４３からなるＣ３Ｅ－７０８８重鎖可変領域及び軽鎖可変領域の組合せ、配列番号５０で表されるアミノ酸番号２～１１９及び１３５～２４３からなるＣ３Ｅ－７０９３重鎖可変領域及び軽鎖可変領域の組合せ、配列番号５４で表されるアミノ酸番号２７２～３８９及び４０５～５１１からなるＣ３Ｅ－７０９６重鎖可変領域及び軽鎖可変領域の組合せ、配列番号５５で表されるアミノ酸番号２７７～３９４及び４１０～５１６からなるＣ３Ｅ－７０９６重鎖可変領域及び軽鎖可変領域の組合せ、配列番号５６で表されるアミノ酸番号２７７～３９４及び４１０～５１６からなるＣ３Ｅ－７０９７重鎖可変領域及び軽鎖可変領域の組合せを含んでなる抗体又はその抗原結合断片を例示することができる。 Further, more preferable antibodies or antigen-binding fragments thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 include the C3E-7034 heavy chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 represented by SEQ ID NO: 46; Combination of light chain variable regions, combination of C3E-7036 heavy chain variable regions and light chain variable regions consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 47, amino acid number 2 represented by SEQ ID NO: 51 A combination of C3E-7078 heavy chain variable region and light chain variable region consisting of ~119 and 135-243, C3E-7085 heavy chain variable region and light chain variable region consisting of amino acid numbers 2-119 and 135-241 represented by SEQ ID NO: 48. Combination of chain variable regions, combination of C3E-7088 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 represented by SEQ ID NO: 49, amino acid numbers 2 to 2 represented by SEQ ID NO: 50 Combination of C3E-7093 heavy chain variable region and light chain variable region consisting of 119 and 135-243, C3E-7096 heavy chain variable region and light chain consisting of amino acid numbers 272-389 and 405-511 represented by SEQ ID NO: 54 Combination of variable regions, combination of C3E-7096 heavy chain variable region and light chain variable region consisting of amino acid numbers 277-394 and 410-516 represented by SEQ ID NO: 55, amino acid numbers 277-394 represented by SEQ ID NO: 56 An example is an antibody or an antigen-binding fragment thereof comprising a combination of the C3E-7097 heavy chain variable region and light chain variable region consisting of 410-516.
　さらに、該ＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含むより好適な抗体又はその抗原結合断片としては、配列番号４６で表されるアミノ酸配列のアミノ酸番号２～２４３のアミノ酸配列からなるＣ３Ｅ－７０３４ｓｃＦｖ、配列番号４７で表されるアミノ酸配列のアミノ酸番号２～２４１のアミノ酸配列からなるＣ３Ｅ－７０３６ｓｃＦｖ、配列番号５１で表されるアミノ酸配列のアミノ酸番号２～２４３のアミノ酸配列からなるＣ３Ｅ－７０７８ｓｃＦｖ、配列番号４８で表されるアミノ酸配列のアミノ酸番号２～２４１のアミノ酸配列からなるＣ３Ｅ－７０８５ｓｃＦｖ、配列番号４９で表されるアミノ酸配列のアミノ酸番号２～２４３のアミノ酸配列からなるＣ３Ｅ－７０８８ｓｃＦｖ、配列番号５０で表されるアミノ酸配列のアミノ酸番号２～２４３のアミノ酸配列からなるＣ３Ｅ－７０９３ｓｃＦｖ、及び、それらのｓｃＦｖのいずれか一つを含む抗体又は抗体の結合断片等を例示することができる。ＣＤ３に特異的なｓｃＦｖ（「抗ＣＤ３　ｓｃＦｖ」とも呼ばれる）の好適な態様にはカルボキシル末端側にＦＬＡＧ－Ｈｉｓタグを付加したもの（単に「タグ付加体」とも呼ばれる）が含まれる。好適なタグ付加体としては、Ｃ３Ｅ－７０３４（配列番号４６）、Ｃ３Ｅ－７０３６（配列番号４７）、Ｃ３Ｅ－７０８５（配列番号４８）、Ｃ３Ｅ－７０８８（配列番号４９）及びＣ３Ｅ－７０９３（配列番号５０）を例示することができ、より好適にはＣ３Ｅ－７０８５をあげることができる。 Furthermore, more preferred antibodies or antigen-binding fragments thereof containing the CDRH1 to CDRH3 and CDRL1 to CDRL3 include C3E-7034scFv, which consists of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence represented by SEQ ID NO: 46; C3E-7036scFv consisting of the amino acid sequence of amino acid numbers 2 to 241 of the amino acid sequence represented by SEQ ID NO. C3E-7085scFv consists of the amino acid sequence of amino acid numbers 2 to 241 of the amino acid sequence represented by SEQ ID NO. 49, C3E-7088scFv consists of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence of SEQ ID NO. Examples include C3E-7093scFv consisting of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence, and antibodies or antibody binding fragments containing any one of these scFvs. Preferred embodiments of CD3-specific scFv (also referred to as "anti-CD3 scFv") include those with a FLAG-His tag added to the carboxyl terminal side (also simply referred to as "tagged product"). Suitable tag adducts include C3E-7034 (SEQ ID NO: 46), C3E-7036 (SEQ ID NO: 47), C3E-7085 (SEQ ID NO: 48), C3E-7088 (SEQ ID NO: 49) and C3E-7093 (SEQ ID NO: 49). 50), and more preferably C3E-7085.
　本発明の多重特異性分子の好適な例として、二重特異性分子を挙げることができる。「二重特異性」とは、同一分子の２つの互いに異なるエピトープ又は２つ分子上の互いに異なるエピトープに結合することが可能であることを意味し、このような二重特異性を有する抗体又は抗原結合断片を包含する。本発明の二重特異性分子は、ＨＬＡ／ＮＹ－ＥＳＯに結合し、さらにＣＤ３に結合する。 A preferred example of the multispecific molecule of the present invention is a bispecific molecule. "Bispecific" means capable of binding to two different epitopes on the same molecule or to different epitopes on two molecules; an antibody or Includes antigen-binding fragments. The bispecific molecules of the invention bind to HLA/NY-ESO and further bind to CD3.
　本発明の二重特異性分子として以下の構造（フォーマット）を有するものが挙げられる。 Examples of the bispecific molecule of the present invention include those having the following structure (format).
　Ｄｕａｌ　ｓｃＦｖ型の二重特異性分子では、異なるエピトープに結合する２種のｓｃＦｖが、二量体のＦｃの一方とそれぞれリンカーにて連結され又はリンカーなしで直接結合されている。あるいは、異なるエピトープに結合する２種類のｓｃＦｖがそれぞれＣＨ、ＣＬにリンカーにて連結され、さらに二量体のＦｃの一方とそれぞれリンカーにて連結されている。該二重特異性分子は、２種類の異なるｓｃＦｖ其々の下流にヘテロダイマーを形成する変異をいれたＦｃがヘテロに会合したフォーマットである。Ｄｕａｌ　ｓｃＦｖ型の二重特異性分子を、Ｄｕａｌ型二重特異性分子、又は単にＤｕａｌ型と呼ぶ（図７(7b))。 In a dual scFv type bispecific molecule, two types of scFv that bind to different epitopes are each linked to one of the dimeric Fcs with a linker or directly linked without a linker. Alternatively, two types of scFv that bind to different epitopes are each linked to CH and CL with a linker, and each is further linked to one of the dimeric Fcs with a linker. The bispecific molecule is in a format in which two different scFvs have heterozygous Fcs containing mutations that form heterodimers downstream of each. A dual scFv type bispecific molecule is called a dual type bispecific molecule or simply a dual type (Figure 7 (7b)).
　本発明においては、例えば、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖと抗ＣＤ３ｓｃＦｖからなるＤｕａｌ型二重特異性分子を用いればよい。 In the present invention, for example, a dual-type bispecific molecule consisting of anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv may be used.
　あるいは、本発明の二重特異性分子として、異なるエピトープに結合するＦａｂとｓｃＦｖが、二量体のＦｃの一方に第１の抗体のＦａｂ、もう一方に第２の抗体のｓｃＦｖを、リンカーを介して結合させた二重特異性分子であってもよい。該二重特異性分子は、Ｆａｂ及びｓｃＦｖのそれぞれ下流にへテロダイマーを形成する変異をいれたＦｃがヘテロに会合したフォーマットである。このような二重特異性分子を、Ｈｙｂｒｉｄ型二重特異性分子、又はＨｙｂｒｉｄ型と呼ぶ（図７(7a)）。本発明においては、例えば、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　Ｆａｂと抗ＣＤ３ｓｃＦｖからなるＨｙｂrｉｄ型を用いることができる。 Alternatively, as a bispecific molecule of the invention, a Fab and scFv that bind to different epitopes are combined with a linker, with the Fab of the first antibody on one side of the dimeric Fc and the scFv of the second antibody on the other side. It may also be a bispecific molecule linked via The bispecific molecule is in a format in which Fab and scFv, each containing a mutation downstream thereof that forms a heterodimer, are assembled in a heterozygous manner. Such a bispecific molecule is called a hybrid type bispecific molecule or a hybrid type (FIG. 7(7a)). In the present invention, for example, a hybrid type consisting of anti-HLA-A2/NY-ESO Fab and anti-CD3 scFv can be used.
　さらに、二量体のＦｃの一方に第１の抗体のＦａｂと第２の抗体のｓｃＦｖを、リンカーを介して結合させた二重特異性分子であってもよい。この場合、ＦｃにＦａｂを結合させ、該ＦａｂにｓｃＦｖを結合させてもよいし、ＦｃにｓｃＦｖを結合させ、該ｓｃＦｖにＦａｂを結合させてもよい。好ましくは、Ｆａｂを結合させ、該ＦａｂにｓｃＦｖを結合させる。ＦａｂとｓｃＦｖの結合は、Ｆａｂの可変領域にリンカーを介してｓｃＦｖを結合させればよい。該二重特異性分子は、ｓｃＦｖとＦａｂをリンカーでつなげた下流にヘテロダイマーを形成する変異をいれたＦｃが会合したフォーマットである。このような二重特異性分子をｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子又はｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型と呼ぶ（図７(７c))。 Furthermore, it may be a bispecific molecule in which the Fab of the first antibody and the scFv of the second antibody are bound to one side of the Fc of the dimer via a linker. In this case, Fab may be bound to Fc and scFv may be bound to the Fab, or scFv may be bound to Fc and Fab may be bound to the scFv. Preferably, a Fab is attached and an scFv is attached to the Fab. Fab and scFv may be linked to the variable region of Fab via a linker. The bispecific molecule has a format in which an scFv and a Fab are connected by a linker, and an Fc containing a mutation that forms a heterodimer is associated downstream of the scFv and Fab. Such a bispecific molecule is called an scFv-Fab-heterodimer Fc-type bispecific molecule or a scFv-Fab-heterodimer Fc-type molecule (FIG. 7(7c)).
　本発明においては、例えば、抗ＣＤ３ｓｃＦｖと抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ＦａｂからなるｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型を用いればよい。 In the present invention, for example, an scFv-Fab-heterodimer Fc type consisting of anti-CD3 scFv and anti-HLA-A2/NY-ESO Fab may be used.
　さらに、第１の抗体と第２の抗体の２種類のｓｃＦｖをリンカーでつなげたフォーマットを有するｔａＦｖ（図５(5c))を、二量体のＦｃの一方にリンカーを介するか又はリンカーを介さずに直接結合させてもよい。このような二重特異性分子をｔａＦｖ－ヘテロダイマーＦｃ型二重特異性分子又はｔａＦｖ－ヘテロダイマーＦｃ型と呼ぶ（図５(5d)）。該二重特異性分子は、ｔａＦｖの下流にヘテロダイマーを形成する変異をいれたＦｃがヘテロに会合したフォーマットである。ｔａＦｖにおける第１の抗体と第２の抗体の連結順序は限定されないが、ｔａＦｖにおける第１の抗体と第２の抗体の連結順序を逆にした場合、最初の二重特異性分子をｔａＦｖ－ヘテロダイマーＦｃ型と呼ぶのに対して、ｔａＦｖ（ｉｎｖｅｒｓｅｄ）－ヘテロダイマーＦｃ型（ｔａＦｖ（ｉｎｖｅｒｓｅｄ）－Ｆｃ型ともいう）と呼ぶ。 Furthermore, taFv (Figure 5 (5c)), which has a format in which two types of scFv, the first antibody and the second antibody, are connected with a linker, is attached to one of the Fc of the dimer via a linker or via a linker. It is also possible to directly combine the two. Such a bispecific molecule is called a taFv-heterodimer Fc type bispecific molecule or a taFv-heterodimer Fc type molecule (FIG. 5(5d)). The bispecific molecule is in a format in which Fc containing a mutation that forms a heterodimer downstream of taFv is heteroassociated. The order of linkage of the first antibody and second antibody in taFv is not limited, but if the order of linkage of the first antibody and second antibody in taFv is reversed, the first bispecific molecule is converted into a taFv-heterogeneous molecule. In contrast to the dimeric Fc type, it is referred to as the taFv(inversed)-heterodimer Fc type (also referred to as the taFv(inversed)-Fc type).
　図７(7a)にＨｙｂｒｉｄ型二重特異性分子、図７(７b)にＤｕａｌ型二重特異的抗体、図７(7c)にｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体の構造を示す。また、図５(5a)にｓｃＦｖ、図５(5b)にＦａｂ、図５(5c)にｔａＦｖ、図５(5d)にｔａＦｖ－ヘテロダイマーＦｃ型二重特異的抗体、図５(5e)にｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体の構造を示す。さらに、図８(８a)にｔａＦｖ－ヘテロダイマーＦｃ型二重特異的抗体（図５(5d)に同じ）、図８(8b)にｔａＦｖ（ｉｎｖｅｒｓｅｄ）－ヘテロダイマーＦｃ型二重特異的抗体、図９(9a)にｔａＦｖ（ｉｎｖｅｒｓｅｄ）－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第１ポリペプチド、図９(9b)にｔａＦｖ（ｉｎｖｅｒｓｅｄ）－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第２ポリペプチドの構造を示す。 Figure 7 (7a) shows the structure of the hybrid type bispecific molecule, Figure 7 (7b) shows the structure of the dual type bispecific antibody, and Figure 7 (7c) shows the structure of the scFv-Fab-heterodimer Fc type bispecific antibody. show. In addition, scFv is shown in Figure 5 (5a), Fab is shown in Figure 5 (5b), taFv is shown in Figure 5 (5c), taFv-heterodimer Fc type bispecific antibody is shown in Figure 5 (5d), and Figure 5 (5e) is The structure of the taFv-Fab-heterodimer Fc type bispecific antibody is shown. Furthermore, Fig. 8 (8a) shows a taFv-heterodimer Fc type bispecific antibody (same as Fig. 5 (5d)), Fig. 8 (8b) shows a taFv (inversed) - heterodimer Fc type bispecific antibody, Figure 9 (9a) shows the first polypeptide contained in the taFv (inversed)-heterodimer Fc type bispecific antibody, and Figure 9 (9b) shows the first polypeptide contained in the taFv (inversed) - heterodimer Fc type bispecific antibody. The structure of the second polypeptide shown in FIG.
　本発明の二重特異的抗体は、複数のポリペプチドが会合した構造を有している。 The bispecific antibody of the present invention has a structure in which multiple polypeptides are associated.
　本発明においては、例えば、ｔａＦｖとしては、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯｓｃＦｖと抗ＣＤ３ｓｃＦｖのｔａＦｖを用いればよい。ｔａＦｖ－ヘテロダイマーＦｃ型二重特異的抗体は、好適には、（ａ）Ｎ末端からＣ末端に向かって、ＨＬＡ／ＮＹ－ＥＳＯに特異的に結合するｓｃＦｖ、ＣＤ３に特異的に結合するｓｃＦｖ及び免疫グロブリンＦｃ領域（ｉ）をその順に含んでなる第１ポリペプチドと、免疫グロブリンのヒンジ領域及びＦｃ領域（ｉｉ）を含んでなる第２ポリペプチドを含み、より好適には、（ｂ）第１ポリペプチドと第２ポリペプチドがＦｃ領域（ｉ）とＦｃ領域（ｉｉ）において会合してなる。第１ポリペプチド及び第２ポリペプチドのＦｃ領域は、ヘテロダイマーを形成するための変異を含んでもよい。ｔａＦｖ－ヘテロダイマーＦｃ型二重特異的抗体の例を図５(5d)に示す。図５(5d)に示すように、第１ポリペプチドのＦｃ領域（ｉ）部分と黒塗で示す第２ポリペプチドのＦｃ領域（ｉｉ）部分が結合し、第１ポリペプチドと第２ポリペプチドが会合している。図６(6a)が第１ポリペプチドを示し、図６(6b)が第２ポリペプチドを示す。例えば、図５(5d)において、白で表されたｓｃＦｖが抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖであり、右上斜線で表されたｓｃＦｖが抗ＣＤ３　ｓｃＦｖである。 In the present invention, for example, anti-HLA-A2/NY-ESOscFv and anti-CD3scFv taFv may be used as the taFv. The taFv-heterodimer Fc type bispecific antibody preferably comprises (a) an scFv that specifically binds to HLA/NY-ESO, an scFv that specifically binds to CD3, from the N-terminus to the C-terminus; and a first polypeptide comprising in that order an immunoglobulin Fc region (i), and a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii), more preferably (b) The first polypeptide and the second polypeptide are associated in Fc region (i) and Fc region (ii). The Fc regions of the first polypeptide and the second polypeptide may include mutations to form a heterodimer. An example of a taFv-heterodimer Fc type bispecific antibody is shown in FIG. 5(5d). As shown in FIG. 5(5d), the Fc region (i) portion of the first polypeptide and the Fc region (ii) portion of the second polypeptide shown in black bind, and the first polypeptide and the second polypeptide are meeting. Figure 6 (6a) shows the first polypeptide, and Figure 6 (6b) shows the second polypeptide. For example, in FIG. 5(5d), the scFv shown in white is anti-HLA-A2/NY-ESO scFv, and the scFv shown with diagonal lines in the upper right corner is anti-CD3 scFv.
　本発明のより好適なｔａＦｖ－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第１ポリペプチドは、配列番号３２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２０番目～５１１番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２０番目～５１６番目のアミノ酸配列、又は配列番号５６で表されるアミノ酸配列の２０番目～５１６番目のアミノ酸配列を含み、より一層好適なｔａＦｖ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第１ポリペプチドは、配列番号３２、３４、３５、３６、３７、３８、３９、４０、４１、４２、４３、３２、５２、５３は５４で表されるアミノ酸配列の５２９番目～７４５番目のアミノ酸配列、あるいは配列番号５５又は５６で表されるアミノ酸配列の５３４番目～７５０番目のアミノ酸配列を含み、さらにより層好適なｔａＦｖ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第１ポリペプチドは、配列番号３２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番４２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列）、配列番号５２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配、列配列番号５３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列又は配列番号５４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列からなる。あるいは、配列番号５５で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列又は配列番号５６で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列からなる。 The first polypeptide contained in the more preferred taFv-heterodimer Fc type bispecific antibody of the present invention has the amino acid sequence from the 21st to the 511th amino acid sequence represented by SEQ ID NO: 32, and the amino acid sequence represented by SEQ ID NO: 34. 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 35, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36 sequence, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, and the amino acid sequence represented by SEQ ID NO: 39. 21st to 511th amino acid sequence, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 42 The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 43, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, Amino acid sequence of 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, amino acid sequence of 21st to 511th of the amino acid sequence represented by SEQ ID NO: 53, amino acid sequence of SEQ ID NO: 54 The 20th to 511th amino acid sequence of the sequence, the 20th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or the 20th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56. The first polypeptide contained in the even more preferred taFv-heterodimer Fc type bispecific molecule includes SEQ ID NOs: 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 32, 52, and 53 include the 529th to 745th amino acid sequence of the amino acid sequence represented by 54, or the 534th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55 or 56, and furthermore, The first polypeptide contained in the preferred taFv-heterodimer Fc-type bispecific molecule has the amino acid sequence from positions 21 to 745 of the amino acid sequence represented by SEQ ID NO: 32, and the amino acid sequence represented by SEQ ID NO: 34. The 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 35, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, SEQ ID NO: The 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40, amino acid sequence of 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 41, represented by SEQ ID NO: 42 (21st to 745th amino acid sequence of the amino acid sequence, 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 43, 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33) , the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, or the amino acid sequence represented by SEQ ID NO: 54. Consists of amino acid sequence from 20th to 745th. Alternatively, it consists of the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55 or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56.
　本発明の好適なｔａＦｖ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第２ポリペプチドは、ヒト抗体由来のヒンジ領域及び変異型Ｆｃを含んでなり、より一層好適なｔａＦｖ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第２ポリペプチドは、配列番号３１で示されるアミノ酸配列の２０番目～２４６番目のアミノ酸配列を含んでなる。 The second polypeptide contained in the preferred taFv-heterodimer Fc-type bispecific molecule of the present invention comprises a human antibody-derived hinge region and a mutant Fc, and is even more preferred taFv-heterodimer Fc-type bispecific molecule. The second polypeptide contained in the bispecific molecule comprises the 20th to 246th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31.
　これらのうち、配列番号３２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００１６、配列番号８４で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００２２、配列番号３５で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００２３、配列番号３６で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００２７、配列番号３７で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００３５、配列番号３８で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００４４、配列番号３９で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００４５、配列番号４０で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００４７、配列番号４１で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００４８、配列番号４２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００６０、配列番号４３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００６１、配列番号３３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００１９、配列番号５２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００１４、並びに、配列番号５３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２１番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＦ－００８２を、本発明の好適なｔａＦｖ－ヘテロダイマーＦｃ型二重特異的抗体として例示することができる。さらに、配列番号５４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２０番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＺ－００３８、配列番号５５で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２０番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＺ－００８２、配列番号５６で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列からなる第１ポリペプチド及び配列番号３１で示されるアミノ酸配列の２０番目～２４６番目からなる第２ポリペプチドが会合してなるＮＹＺ－００８３を、本発明の好適なｔａＦｖ－ヘテロダイマーＦｃ型二重特異的抗体として例示することができる。 Among these, the first polypeptide consists of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, and the second polypeptide consists of the 21st to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 31. NYF-0016 formed by association, a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 84, and a first polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31. NYF-0022, which is formed by association of two polypeptides, a first polypeptide consisting of the amino acid sequence 21st to 745th of the amino acid sequence represented by SEQ ID NO: 35, and a first polypeptide consisting of the amino acid sequence 21st to 246th of the amino acid sequence represented by SEQ ID NO: 31. NYF-0023, which is composed of a second polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 36, a first polypeptide consisting of the amino acid sequence 21st to 745th of the amino acid sequence represented by SEQ ID NO: 36, and a first polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 31. NYF-0027 formed by association of a second polypeptide consisting of positions 21 to 246, a first polypeptide consisting of the amino acid sequence 21 to 745 of the amino acid sequence represented by SEQ ID NO: 37, and a first polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 31 NYF-0035, which is a combination of a second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, and a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38; NYF-0044, which is formed by combining the second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence shown by number 31, and the first consisting of the 21st to 745th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 39. NYF-0045, which is a combination of a polypeptide and a second polypeptide consisting of positions 21 to 246 of the amino acid sequence represented by SEQ ID NO: 31, an amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO: 40; NYF-0047 is a combination of a first polypeptide consisting of a first polypeptide consisting of a polypeptide consisting of a second polypeptide consisting of positions 21 to 246 of the amino acid sequence shown in SEQ ID NO: 31, and a second polypeptide consisting of positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 41. NYF-0048, which is a combination of a first polypeptide consisting of the amino acid sequence of No. 21 and a second polypeptide consisting of the 21st to 246th amino acid sequence of SEQ ID NO: 31, has an amino acid sequence of SEQ ID NO: 42. NYF-0060, which is a combination of a first polypeptide consisting of the 21st to 745th amino acid sequence and a second polypeptide consisting of the 21st to 246th amino acid sequence of SEQ ID NO: 31, represented by SEQ ID NO: 43; NYF-0061, which is formed by an association of a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31 and a second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31, NYF-, which is formed by association of a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33 and a second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31. 0019, a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52 and a second polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31 are associated. a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, and a first polypeptide consisting of the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31. NYF-0082, which is formed by association of two polypeptides, can be exemplified as a preferred taFv-heterodimer Fc type bispecific antibody of the present invention. Furthermore, a first polypeptide consisting of the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54 and a second polypeptide consisting of the 20th to 246th amino acid sequence of SEQ ID NO: 31 are associated. NYZ-0038, a first polypeptide consisting of the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, and a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31. NYZ-0082 formed by association of peptides, a first polypeptide consisting of the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, and the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31. NYZ-0083, which is formed by association of a second polypeptide, can be exemplified as a preferred taFv-heterodimer Fc type bispecific antibody of the present invention.
　それらのうち、ＮＹＦ－００２３、ＮＹＦ－００４７、ＮＹＦ－００４８、ＮＹＦ－００６０、ＮＹＦ－００６１、ＮＹＺ－００３８、ＮＹＺ－００８２、及びＮＹＺ－００８３は、とりわけ優れた生物活性、物性等を有し、好適である。 Among them, NYF-0023, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, and NYZ-0083 have particularly excellent biological activity, physical properties, etc. suitable.
　さらに、二量体のＦｃの一方に第１の抗体のｔａＦｖを、リンカーを介するか又はリンカーを介さずに直接結合させ、もう一方のＦｃに第１の抗体若しくは第２の抗体のＦａｂを、リンカーを介するか又はリンカーを介さずに直接結合させてもよい。該二重特異性分子は、ｔａＦｖ－ヘテロダイマーＦｃ型の第２ポリペプチドのＦｃ領域（ｉｉ）(黒塗り)側の上流にＦａｂを付加したフォーマットである。このような二重特異性分子をｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子又はｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型と呼ぶ（図５(5e)）。 Furthermore, the taFv of the first antibody is directly bound to one of the Fc of the dimer, with or without a linker, and the Fab of the first antibody or the second antibody is bound to the other Fc, They may be linked directly through a linker or without a linker. The bispecific molecule has a format in which Fab is added upstream of the Fc region (ii) (blacked out) side of the taFv-heterodimer Fc type second polypeptide. Such a bispecific molecule is called a taFv-Fab-heterodimer Fc-type bispecific molecule or a taFv-Fab-heterodimer Fc-type molecule (FIG. 5(5e)).
　本発明においては、ｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子に含まれるｔａＦｖとしては、例えば、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ ｓｃＦｖと抗ＣＤ３ｓｃＦｖのｔａＦｖを用いればよく、Ｆａｂとしては、例えば、ＨＬＡ／ＮＹ－ＥＳＯ　Ｆａｂを用いればよい。 In the present invention, as the taFv contained in the taFv-Fab-heterodimer Fc type bispecific molecule, for example, anti-HLA-A2/NY-ESO scFv and anti-CD3 scFv taFv may be used, and as the Fab, For example, HLA/NY-ESO Fab may be used.
　ｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体は、好適には、（ａ）Ｎ末端からＣ末端に向かって、ヒトＨＬＡ／ＮＹ－ＥＳＯに特異的に結合するｓｃＦｖ、ＣＤ３に特異的に結合するｓｃＦｖ及び免疫グロブリンＦｃ領域（ｉ）をその順で含んでなる第１ポリペプチド、Ｆｃ領域（ｉｉ）を含む免疫グロブリン重鎖からなる第２ポリペプチド、及び、免疫グロブリン軽鎖からなる第３ポリペプチドを含み、より好適には、（ｂ）第２ポリペプチドと第３ポリペプチドが会合しており、（ｃ）第１ポリペプチドと第２ポリペプチドがＦｃ領域（ｉ）とＦｃ領域（ｉｉ）において会合してなる。ｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体の例を図５(5e）に示し、第１ポリペプチドを図６(6a）に示し、第２ポリペプチドを図６(6c）に示し、第３ポリペプチドを図６（6d）に示す。図５(5e）に示すように、第１ポリペプチドのＦｃ領域（ｉ）部分と黒塗で示すＦｃ領域（ｉｉ）を含む免疫グロブリン重鎖からなる第２ポリペプチドが第１ポリペプチドのＦｃ領域（ｉ）部分と第２ポリペプチドのＦｃ領域（ｉｉ）部分で結合し、さらに、第２ポリペプチドに免疫グロブリン軽鎖が結合している。かかる好適なｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子は、ｔａＦｖ及び免疫グロブリンのＦｃ領域を含むｔａＦｖ－ヘテロダイマーＦｃ型二重特異性分子に、Ｆａｂが結合してなる、ということもできる。好適なｔａＦｖ－ＦａｂヘテロダイマーＦｃ型二重特異性分子に含まれる第１ポリペプチドに含まれるアミノ酸配列としては、配列番号３２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列、及び、配列番号５６で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列を例示することができ、さらに好適なｔａＦｖ－ＦａｂヘテロダイマーＦｃ型二重特異性分子に含まれる第１ポリペプチドは、配列番号３２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列及び配列番号３３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２１番目～７４５番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２１番目～７５０番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２１番目～７５０番目のアミノ酸配列からなる。 The taFv-Fab-heterodimer Fc type bispecific antibody preferably comprises (a) from the N-terminus toward the C-terminus, an scFv that specifically binds to human HLA/NY-ESO, and a scFv that specifically binds to CD3. a first polypeptide comprising a binding scFv and an immunoglobulin Fc region (i) in that order; a second polypeptide comprising an immunoglobulin heavy chain comprising an Fc region (ii); and a second polypeptide comprising an immunoglobulin light chain. more preferably, (b) the second polypeptide and the third polypeptide are associated, and (c) the first polypeptide and the second polypeptide are associated with the Fc region (i) and the Fc region. (ii). An example of a taFv-Fab-heterodimer Fc type bispecific antibody is shown in FIG. 5(5e), the first polypeptide is shown in FIG. 6(6a), the second polypeptide is shown in FIG. 6(6c), The third polypeptide is shown in Figure 6 (6d). As shown in FIG. 5(5e), the second polypeptide consisting of an immunoglobulin heavy chain including the Fc region (i) portion of the first polypeptide and the Fc region (ii) shown in black is connected to the Fc region (i) of the first polypeptide. The region (i) portion is bound to the Fc region (ii) portion of the second polypeptide, and further, an immunoglobulin light chain is bound to the second polypeptide. Such a preferred taFv-Fab-heterodimer Fc-type bispecific molecule is formed by binding Fab to a taFv-heterodimer Fc-type bispecific molecule comprising taFv and immunoglobulin Fc regions. can. The amino acid sequence contained in the first polypeptide contained in the preferred taFv-Fab heterodimer Fc type bispecific molecule includes the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, SEQ ID NO: The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, and the amino acid sequence represented by SEQ ID NO: 39 The 21st to 511th amino acid sequence of the amino acid sequence, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 41, The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 42, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 43, the 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33 ~511th amino acid sequence, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, SEQ ID NO: 54 the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, and the 21st to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56. The first polypeptide contained in the more preferable taFv-Fab heterodimer Fc type bispecific molecule is the amino acid sequence from the 21st to the 745th amino acid sequence represented by SEQ ID NO: 32. Amino acid sequence, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequence represented by SEQ ID NO: 36 The 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, SEQ ID NO: The 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 42, 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 43, and the amino acid sequence represented by SEQ ID NO: 33. The 21st to 745th amino acid sequence of the amino acid sequence, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, The 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54, the 21st to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or the amino acid sequence represented by SEQ ID NO: 56. Consists of amino acid sequence 21st to 750th.
　本発明の好適なｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第２ポリペプチドは、ヒト抗体又はヒト化抗体重鎖の可変領域、ＣＨ１領域及びヒンジ領域、並びに変異型Ｆｃを含んでなり、より好適なｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第２ポリペプチドは、配列番号４４で表されるアミノ酸配列の２０番目～２４２番目のアミノ酸配列を含んでなる。 The second polypeptide contained in the preferred taFv-Fab-heterodimer Fc type bispecific antibody of the present invention comprises the variable region, CH1 region and hinge region of a human antibody or humanized antibody heavy chain, as well as mutant Fc. The second polypeptide contained in the more preferred taFv-Fab-heterodimer Fc type bispecific antibody comprises the 20th to 242nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 44. Become.
　本発明の好適なｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第３ポリペプチドは、ヒト抗体又はヒト化抗体軽鎖の可変領域及び定常領域を含んでなり、より好適なｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第３ポリペプチドは、配列番号４５で表されるアミノ酸配列の２１番目～１３１番目を含んでなる。 The third polypeptide contained in the preferred taFv-Fab-heterodimer Fc type bispecific antibody of the present invention comprises the variable region and constant region of a human antibody or humanized antibody light chain, and is a more preferred taFv The third polypeptide contained in the -Fab-heterodimer Fc type bispecific molecule comprises the 21st to 131st amino acid sequences represented by SEQ ID NO: 45.
　かかる好適なｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第２ポリペプチドのうち可変領域及びＣＨ１領域並びに第３ポリペプチドはＦａｂを構成し、好適なＦａｂは抗ＨＬＡ／ＮＹ－ＥＳＯ抗体のＦａｂであり、例えば、ＮＹＡ－０００１のＦａｂである。 The variable region and CH1 region of the second polypeptide contained in this preferred taFv-Fab-heterodimer Fc type bispecific molecule and the third polypeptide constitute Fab, and the preferred Fab is anti-HLA/NY- Fab of ESO antibody, for example, Fab of NYA-0001.
　本発明においては、ｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体に含まれるｓｃＦｖとしては、例えば、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ ｓｃＦｖ又は抗ＣＤ３ｓｃＦｖを用いればよく、Ｆａｂとしては、例えば、ＨＬＡ／ＮＹ－ＥＳＯ　Ｆａｂ又は抗ＣＤ３　Ｆａｂを用いればよい。 In the present invention, as the scFv included in the scFv-Fab-heterodimer Fc type bispecific antibody, for example, anti-HLA-A2/NY-ESO scFv or anti-CD3 scFv may be used, and as the Fab, for example, HLA/NY-ESO Fab or anti-CD3 Fab may be used.
　ｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子は、好適には、（ａ）Ｎ末端からＣ末端に向かって、ヒトＨＬＡ／ＮＹ－ＥＳＯに特異的に結合するｓｃＦｖ、ＣＤ３に特異的に結合する抗体重鎖の可変領域及び定常領域ＣＨ１並びに免疫グロブリンＦｃ領域（ｉ）をその順で含んでなる第１ポリペプチド、免疫グロブリンのヒンジ領域及びＦｃ領域（ｉｉ）を含んでなる第２ポリペプチド、並びに、可変領域及び定常領域からなる抗体軽鎖を含む第３ポリペプチドを含み、より好適には、（ｂ）第１ポリペプチドと第２ポリペプチドがＦｃ領域（ｉ）とＦｃ領域（ｉｉ）において会合し、第１ポリペプチドが抗体重鎖の可変領域及び定常領域ＣＨ１において第３ポリペプチド（の抗体軽鎖）と会合してなる。第１ポリペプチド及び第２ポリペプチドのＦｃ領域は、野生型であっても、ヘテロダイマーを形成する変異を含んでいてもよい。ｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子の例を図７（7ｃ）に示す。図７（7ｃ）の右半分が第１ポリペプチド及び第３ポリペプチドであり、左半分が第２ポリペプチドである。図７（７ｃ）に示すように、第１ポリペプチドのＦｃ領域（ｉ）部分と黒塗で示す第２ポリペプチドのＦｃ領域（ｉｉ）部分が会合し、第１ポリペプチドと第３ポリペプチドが会合している。例えば、図７(7c)において、右上斜線で表されたｓｃＦｖが抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ　ｓｃＦｖであり、白及び市松模様及び横線で表されたＦａｂが抗ＣＤ３　Ｆａｂである。 The scFv-Fab-heterodimer Fc-type bispecific molecule preferably comprises (a) from the N-terminus towards the C-terminus, an scFv that specifically binds to human HLA/NY-ESO, and a scFv that specifically binds to CD3; A first polypeptide comprising, in that order, the variable region and constant region CH1 of an antibody heavy chain to be bound and an immunoglobulin Fc region (i), and a second polypeptide comprising an immunoglobulin hinge region and Fc region (ii). peptide and a third polypeptide comprising an antibody light chain consisting of a variable region and a constant region; more preferably, (b) the first polypeptide and the second polypeptide have an Fc region (i) and an Fc region ( ii), the first polypeptide is associated with the third polypeptide (the antibody light chain) in the variable region and constant region CH1 of the antibody heavy chain. The Fc regions of the first polypeptide and the second polypeptide may be wild type or may contain mutations that form a heterodimer. An example of a scFv-Fab-heterodimer Fc type bispecific molecule is shown in Figure 7 (7c). The right half of FIG. 7 (7c) is the first polypeptide and the third polypeptide, and the left half is the second polypeptide. As shown in FIG. 7 (7c), the Fc region (i) portion of the first polypeptide and the Fc region (ii) portion of the second polypeptide shown in black associate with each other, and the first polypeptide and the third polypeptide are meeting. For example, in FIG. 7(7c), the scFv indicated by diagonal lines in the upper right is anti-HLA-A2/NY-ESO scFv, and the Fab indicated by white, checkered patterns, and horizontal lines is anti-CD3 Fab.
　好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第１ポリペプチドに含まれるアミノ酸配列としては、配列番号５７で表されるアミノ酸配列の２１番目～３９４番目のアミノ酸配列を、さらに好適には２０番目～７２４番目のアミノ酸配列を、それぞれ例示することができる。 The amino acid sequence contained in the first polypeptide contained in the preferred scFv-Fab-heterodimer Fc type bispecific antibody is the 21st to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 57, More preferably, the 20th to 724th amino acid sequences can be exemplified.
　また、好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第１ポリペプチドに含まれる他のアミノ酸配列としては、配列番号６０で表されるアミノ酸配列の２１番目～３８９番目のアミノ酸配列を、さらに好適には２０番目～７１９番目のアミノ酸配列を、それぞれ例示することができる。 In addition, other amino acid sequences contained in the first polypeptide contained in the preferred scFv-Fab-heterodimer Fc type bispecific antibody include the 21st to 389th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 60. The amino acid sequence, more preferably the 20th to 719th amino acid sequence, can be exemplified.
　さらに、好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第１ポリペプチドに含まれる他のアミノ酸配列としては、配列番号６１で表されるアミノ酸配列の２１番目～３８９番目のアミノ酸配列を、さらに好適には２０番目～７１９番目のアミノ酸配列を、それぞれ例示することができる。 Furthermore, other amino acid sequences contained in the first polypeptide contained in the preferred scFv-Fab-heterodimer Fc type bispecific antibody include the 21st to 389th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 61. The amino acid sequence, more preferably the 20th to 719th amino acid sequence, can be exemplified.
　本発明の好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異的抗体に含まれる第２ポリペプチドは、ヒト抗体由来のヒンジ領域及び変異型Ｆｃを含んでなり、より一層好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第２ポリペプチドは、配列番号３１で示されるアミノ酸配列の２０番目～２４６番目のアミノ酸配列を含んでなる。 The second polypeptide contained in the preferred scFv-Fab-heterodimer Fc type bispecific antibody of the present invention comprises a hinge region derived from a human antibody and a mutant Fc, and an even more preferred scFv-Fab- The second polypeptide contained in the heterodimer Fc type bispecific molecule comprises the 20th to 246th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31.
　本発明の好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第３ポリペプチドは、ヒト抗体由来の軽鎖を含んでなり、より好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第３ポリペプチドとしては、配列番号５８で示されるアミノ酸配列の２１番目～１２７番目のアミノ酸配列、より一層好適には２１番目～２３３番目のアミノ酸配列を、それぞれ例示することができる。 The third polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a light chain derived from a human antibody, and the third polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule comprises Examples of the third polypeptide included in the heavy specificity molecule include the 21st to 127th amino acid sequence, more preferably the 21st to 233rd amino acid sequence of the amino acid sequence shown by SEQ ID NO: 58. be able to.
　これらのうち、配列番号５７で表されるアミノ酸配列の２０番目～７２４番目のアミノ酸配列からなる第１ポリペプチド、配列番号３1で示されるアミノ酸配列の２０番目～２４６番目からなる第２ポリペプチド、及び、配列番号５８で示されるアミノ酸配列の２１番目～２３３番目からなる第３のポリペプチドが会合してなるＮＹＺ－１０１０を、本発明の好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子として例示することができる。 Among these, a first polypeptide consisting of the 20th to 724th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 57, a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31, And NYZ-1010, which is formed by association of a third polypeptide consisting of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 58, is used as a preferred scFv-Fab-heterodimer Fc type bispecific of the present invention. It can be exemplified as a molecule.
　また、配列番号６０で表されるアミノ酸配列の２０番目～７１９番目のアミノ酸配列からなる第１ポリペプチド、配列番号３１で示されるアミノ酸配列の２０番目～２４６番目からなる第２ポリペプチド、及び、配列番号５８で示されるアミノ酸配列の２１番目～２３３番目からなる第３のポリペプチドが会合してなるＮＹＺ－１００７も、本発明の好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子として例示することができる。 Further, a first polypeptide consisting of the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 60, a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31, and NYZ-1007, which is formed by association of a third polypeptide consisting of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 58, is also suitable as a preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention. I can give an example.
　さらに、配列番号６１で表されるアミノ酸配列の２０番目～７１９番目のアミノ酸配列からなる第１ポリペプチド、配列番号３１で示されるアミノ酸配列の２０番目～２４６番目からなる第２ポリペプチド、及び、配列番号５８で示されるアミノ酸配列の２１番目～２３３番目からなる第３のポリペプチドが会合してなるＮＹＺ－１０１７も、本発明の好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子として例示することができる。 Furthermore, a first polypeptide consisting of the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 61, a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31, and NYZ-1017, which is formed by association of a third polypeptide consisting of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 58, is also suitable as a preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention. I can give an example.
　本発明の二重特異性分子に含まれる１つ、２つ又は３つ以上のペプチドは、前述の「欠失体」であってよく、すなわち、そのカルボキシル末端、とりわけ抗体重鎖に由来するカルボキシル末端において、１又は２個（又はそれ以上）のアミノ酸が変異（欠失を含む）していてよい。例えば、本発明の好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子の一つであるＮＹＺ－１０１０に含まれる第１ポリペプチドが有するアミノ酸配列のカルボキシル末端は、配列番号５７の７２４番目Ｌｙｓ、１アミノ酸が欠失してなる７２３番目Ｇｌｙ、それらを含む混合物、のいずれであってもよい。同様に、本発明の好適なｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型二重特異性分子に含まれる第２ポリペプチドが有するアミノ酸配列のカルボキシル末端は、配列番号８４の２４６番目Ｌｙｓ、１アミノ酸が欠失してなる２４５番目Ｇｌｙ、それらを含む混合物、のいずれであってもよい。 One, two or more of the peptides comprised in the bispecific molecule of the invention may be "deletions" as described above, i.e. at their carboxyl terminus, especially at the carboxyl end derived from the antibody heavy chain. At the terminus, one or two (or more) amino acids may be mutated (including deletions). For example, the carboxyl terminal of the first polypeptide contained in NYZ-1010, which is one of the preferred scFv-Fab-heterodimer Fc type bispecific molecules of the present invention, is the 724th amino acid sequence of SEQ ID NO: 57. It may be Lys, Gly at position 723 with one amino acid deleted, or a mixture containing them. Similarly, the carboxyl terminal of the amino acid sequence of the second polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention has one amino acid deletion at Lys at position 246 of SEQ ID NO: 84. or a mixture containing them.
　本発明の二重特異性分子に含まれるｓｃＦｖ及びＦａｂは、好ましくは、ヒト化抗体又はヒト抗体のｓｃＦｖ及びＦａｂであり、Ｆｃは、好ましくは、ヒト抗体のＦｃである。 The scFv and Fab contained in the bispecific molecule of the present invention are preferably those of a humanized antibody or a human antibody, and the Fc is preferably that of a human antibody.
　本発明の二重特異性分子に含まれる可変領域においては、抗体のアミノ末端側から重鎖可変領域、軽鎖可変領域の順で結合していてもよく、あるいは、軽鎖可変領域、重鎖可変領域の順で結合していてもよい。両可変領域の間に、リンカーを有していてもよい（任意で）。また、アミノ末端側の可変領域のアミノ末端にグリシン残基を有していてもよい（任意で）。タンデムｓｃＦｖ型の二重特異性分子では、カルボキシル末端側の可変領域のカルボキシル末端に、リンカー、ＦＬＡＧタグ、及び／又は、Ｈｉｓタグが結合していてもよい（任意で）。好適な態様の一つとして、アミノ末端から、重鎖可変領域、第１のリンカー、軽鎖可変領域、第２のリンカー、ＦＬＡＧタグ、及び、Ｈｉｓタグの順に結合したものを例示することができる。 In the variable regions contained in the bispecific molecule of the present invention, the heavy chain variable region and the light chain variable region may be linked in this order from the amino terminal side of the antibody, or the light chain variable region and the heavy chain variable region may be linked in this order from the amino terminal side of the antibody. They may be linked in the order of variable regions. A linker may be present between both variable regions (optionally). The variable region may also have a glycine residue at the amino terminus (optionally). In tandem scFv-type bispecific molecules, a linker, a FLAG tag, and/or a His tag may (optionally) be attached to the carboxyl terminus of the variable region on the carboxyl terminal side. One preferred embodiment is one in which the heavy chain variable region, the first linker, the light chain variable region, the second linker, the FLAG tag, and the His tag are linked in this order from the amino terminal. .
　リンカーは、一本鎖ポリペプチド又は一本鎖オリゴペプチド、あるいは、ＰＥＧ、ヌクレオチド、糖鎖、化合物等の合成品も含まれる。その他、二つのポリペプチドを結合するものであれば特に限定されず、公知のリンカーを使用することが可能である。 Linkers include single-chain polypeptides or single-chain oligopeptides, or synthetic products such as PEG, nucleotides, sugar chains, and compounds. In addition, any known linker can be used without particular limitation as long as it connects two polypeptides.
　リンカーの長さとしては、例えば、ペプチドリンカーの場合５～３０アミノ酸である。二重特異性分子に複数のリンカーが含まれる場合、すべて同じ長さのペプチドリンカーを用いてもよいし、異なる長さのペプチドリンカーを用いてもよい。 The length of the linker is, for example, 5 to 30 amino acids in the case of a peptide linker. If the bispecific molecule includes multiple linkers, all peptide linkers may be of the same length, or peptide linkers of different lengths may be used.
　ペプチドリンカーとしては、例えば、（Ｇｌｙ・Ｇｌｙ・Ｇｌｙ・Ｇｌｙ・Ｓｅｒ）の繰り返しが例示されるが、これらに１～数個のＧｌｙ、Ｓｅｒとは異なるアミノ酸残基が付加していてもよい。 Examples of the peptide linker include repeats of (Gly・Gly・Gly・Gly・Ser), but one to several amino acid residues different from Gly and Ser may be added to these.
　上述のような、本発明の多重特異性抗体、特に二重特異性抗体が採り得る構造（フォーマット）のうち、好適なものは、ｔａＦｖ－ヘテロダイマーＦｃ型、ｔａＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型及びｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型であり、より好適なものはｔａＦｖ－ヘテロダイマーＦｃ型であり、Ｎ末端からＣ末端に向かって、抗ＨＬＡ／ＮＹ－ＥＳＯ　ｓｃＦｖ、抗ＣＤ３　ｓｃＦｖの順に位置している型（ｔａＦｖ－ヘテロダイマーＦｃ型）は、その逆に位置している型（ｔａＦｖ（ｉｎｖｅｒｓｅｄ）－ヘテロダイマーＦｃ型）に比してより一層好適である。また、他のより好適なものはｓｃＦｖ－Ｆａｂ－ヘテロダイマーＦｃ型である。 Among the structures (formats) that can be adopted by the multispecific antibody, particularly the bispecific antibody, of the present invention as described above, preferred ones are taFv-heterodimer Fc type, taFv-Fab-heterodimer Fc type, and The scFv-Fab-heterodimer Fc type is preferred, and the more preferable one is the taFv-heterodimer Fc type, which is located in the order of anti-HLA/NY-ESO scFv and anti-CD3 scFv from the N-terminus to the C-terminus. The inverted type (taFv-heterodimer Fc type) is more suitable than the opposite type (taFv (inverted)-heterodimer Fc type). Another more preferred type is the scFv-Fab-heterodimer Fc type.
　本発明には、本発明の分子に含まれるアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチドの相補鎖とストリンジェントな条件下でハイブリダイズするポリヌクレオチドに含まれるヌクレオチド配列によりコードされるアミノ酸配列を含み、且つ、ＨＬＡ／ＮＹ－ＥＳＯに結合し、好適にはさらにＣＤ３にも結合する分子も含まれる。 The present invention includes an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence contained in the molecule of the present invention. Also included are molecules that contain and bind to HLA/NY-ESO and preferably also bind to CD3.
　本発明には、本発明の分子に含まれるアミノ酸配列と９０％、９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％、又は９９％以上同一のアミノ酸配列を含み、且つ、ＨＬＡ／ＮＹ－ＥＳＯに結合し、好適にはさらにＣＤ３にも結合する分子も含まれる。 The present invention includes amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to the amino acid sequences contained in the molecules of the present invention. and binds to HLA/NY-ESO and preferably also binds to CD3.
　本発明の抗体、その結合断片及びそれらを含む多重特異性抗体は、優れた生物学的活性、物理化学的性質（以下「物性」という。）、安全性、体内動態等の性質を具備している。（ａ）生物学的活性又はその指標としては、抗原結合活性、ｉｎ　ｖｉｔｒｏ細胞傷害活性、ｉｎ　ｖｉｖｏ抗腫瘍活性等を例示することができる。例えば、ＨＬＡ／ＮＹ－ＥＳＯに対する解離定数（ＫＤ値）は、１００ｎＭ以下又は５０ｎＭ以下、好適には２０ｎＭ以下又は１０ｎＭ以下、より好適には５ｎＭ以下である。また、例えば、内因性ヒトＮＹ－ＥＳＯ発現細胞株であるＵ２６６Ｂ１及び／又はＮＣＩ－Ｈ１７０３に対して、ヒト末梢血単核細胞をエフェクター細胞として用いて発揮される細胞傷害活性のＥＣ５０値は２０ｎＭ以下、好適には１０ｎＭ以下、より好適には５ｎＭ以下である（ｉｎ　ｖｉｔｒｏ細胞傷害活性の測定及び算出としては、ＷＯ２０２１／２００８５７の実施例８記載の方法を例示できるが、それに限定されない）。さらに、例えば、ヒト扁平上皮肺がん細胞株ＮＣＩ－Ｈ１７０３の６×１０７細胞／ｍＬ懸濁液をＮＯＧマウスの皮下に０．１ｍＬ移植し、４日後ヒト末梢血単核細胞の３．７５×１０７細胞／ｍＬ懸濁液を０．２ｍＬ尾静脈内移植し、１４日後から週１回、抗体を３回投与後腫瘍の体積を測定した際の、溶媒を投与した対照群に対する腫瘍増殖阻害活性は、５０％以上、好適には７５％以上、より好適には９０％以上である（ｉｎ　ｖｉｖｏ抗腫瘍活性の測定及び算出としては、実施例９記載の方法を例示できるが、それに限定されない）。（ｂ）バイオ医薬品に含まれる不純物は、医薬品の安全性に関与するため、製造時および保存中での増加の有無を適切に規格設定し管理することが求められている。その中でもＨＭＷＳ（凝集物）は、主要な不純物の一つであり、免疫原性リスクや薬効の低下等にも関与するため、特に厳重に管理すべき項目である。不純物の管理は製造時のみでなく、製造工程中や製造後の経時的な安定性（増加の有無）も含めて評価を実施する必要がある。長期安定性試験の結果に基づいて医薬品の有効期間が設定されるため、経時的に安定な抗体のほうがより長い有効期間を設定することができる。よってバイオ医薬品をめざし好適な抗体を選抜する際の指標となる、本発明における物理化学的性質としては、耐酸性（ＨＭＷＳ生成抑制等）、溶液安定性（ＨＭＷＳ生成抑制等）が挙げられる。また他の指標としては、本発明の抗体若しくはその結合断片又はそれらを含む分子の産生に適した宿主細胞、例えば、Ｅｘｐｉ２９３Ｆ細胞にそれらに含まれるアミノ酸配列をコードする遺伝子を導入して得られる組換え細胞の培養物における収量が多い、収率が高いことなどを例示することができる。それらの物理化学的性質特徴を有する本発明の好適な抗体、その抗原結合断片、及び、それらを含有する多重特異性抗体は：それらの溶液を酸性条件下に晒すことが可能となり、それらの製造、例えば、プロテインＡ、イオン交換等のクロマトグラフィー、ウイルス不活性化等の工程の実施が可能又は容易になる；それらを溶液としてもＨＭＷＳの生成が低く抑えられるので、それらの製造、製剤化、それらを含む医薬品の流通や保存等の実施が可能又は容易になる；それらを効率よく生産することが可能となる。耐酸性について、例えば、ｐＨ３．５、室温、１時間保温し、次いでサイズ排除クロマトグラフィーによりＨＭＷＳを測定して算出される生じるＨＭＷＳの含有量は５％以下、好適には２％以下、より好適には１％以下である（耐酸性評価のためのＨＭＷＳ含有量の測定及び算出としては、ＷＯ２０２１／２００８５７の実施例１９－１記載の方法を例示できるが、それに限定されない）。溶液安定性について、例えば、２５ｍＭヒスチジン及び５％ソルビトールからなるｐＨ６．０に濃度２５ｍｇ/ｍｌになるよう溶解させ、２５℃、６日間保存し、次いでサイズ排除クロマトグラフィーによりＨＭＷＳを測定して算出されるＨＭＷＳの含有量は２０％以下、好適には１０％以下である（溶液安定性評価のためのＨＭＷＳ含有量の測定及び算出としては、ＷＯ２０２１／２００８５７の実施例１９－２記載の方法を例示できるが、それに限定されない）。生産効率又は収率の測定及び算出としては、ＷＯ２０２１／２００８５７の実施例２０及び２１記載の方法を例示できるが、それに限定されない。（ｃ）安全性又はその指標としては、抗原認識特性、投与時の所見等を例示することができる。例えば、野生型ＮＹ－ＥＳＯペプチド上の複数のアミノ酸を認識し、野生型ＮＹ－ＥＳＯペプチドに類似するが同一ではないアミノ酸配列を有する相同性ペプチドには結合せず、オフターゲット効果に起因する副作用リスクが低い。また、ＩＳＰＲＩ ウェブベース免疫原性スクリーニング（ＥｐｉＶａｘ，Ｉｎｃ）において免疫原性が低く、抗抗体に起因するサイトカイン産生等の副作用リスクが低いことが予測できる。また、例えば、本発明の二重特異性抗体に含まれるＮＹＦ－００２３、ＮＹＦ－００４５、ＮＹＦ－００４７、ＮＹＦ－００４８、ＮＹＦ－００６０、ＮＹＦ－００６１、ＮＹＺ－００８２、又は、ＮＹＺ－１０１０をＢａｌｂ／ｃマウスに投与したところ、血中半減期には問題となるような所見は認められず、体重減少、その他の顕著な毒性所見も認められなかった。また、ＮＹＺ－００８２、又は、ＮＹＺ－１０１０をカニクイザルに単回投与したところ、血中半減期には問題となるような所見は認められず、一般状態、体重、摂餌量、体温測定、及び血漿中サイトカイン量について投与に起因する変化は認められなかった。（ｄ）動態又はその指標としては、血中半減期等を例示することができる。例えば、本発明において取得した数個の二重特異性抗体をＢａｌｂ／ｃマウス、又は、カニクイザルに投与したところ、血中半減期には問題となるような所見は認められなかった。このような、優れた生物学的活性、物理化学的性質、安全性、体内動態等の性質を具備する本発明の抗体、その結合断片及び分子は、医薬組成物に好適に含有せしめることができる。（ａ）記載の抗原結合活性及び（ｂ）記載の諸性質を有する本発明の好適な抗体又はその抗原結合断片としては、ＮＹＡ－１１４３，ＮＹＡ－１１６３、ＮＹＡ－２０２３、ＮＹＡ－２０２７、ＮＹＡ－２０３５、ＮＹＡ－２０４４，ＮＹＡ－２０４５、ＮＹＡ－２０４７、ＮＹＡ－２０４８、ＮＹＡ－２０６０、ＮＹＡ－２０３１、ＮＹＡ－２０４７、ＮＹＡ－２０６１、ＮＹＡ－２１４３及びＮＹＡ－３０６１を、より好適にはＮＹＡ－２０４７、ＮＹＡ－２０６１、ＮＹＡ－２１４３及びＮＹＡ－３０６１を、それぞれ例示することができるが、それらに限定されない。また、（ａ）～（ｄ）記載の性質を有する本発明の好適な多重特異性抗体としては、ＮＹＦ－００１６、ＮＹＦ－００１９、ＮＹＦ－００２２、ＮＹＦ－００２３、ＮＹＦ－００２７、ＮＹＦ－００３５、ＮＹＦ－００４４、ＮＹＦ－００４５、ＮＹＦ－００４７、ＮＹＦ－００４８、ＮＹＦ－００５８、ＮＹＦ－００６０、ＮＹＦ－００６１、ＮＹＺ－００３８、ＮＹＺ－００８２、ＮＹＺ－００８８及びＮＹＺ－１０１０を、より好適には、ＮＹＦ－００６１、ＮＹＺ－００３８、ＮＹＺ－００８２、ＮＹＺ－００８８、ＮＹＺ－１００７、ＮＹＺ－１０１０、ＮＹＺ－１０１７を、それぞれ例示することができるが、それらに限定されない。 The antibodies of the present invention, binding fragments thereof, and multispecific antibodies containing them have excellent biological activity, physicochemical properties (hereinafter referred to as "physical properties"), safety, pharmacokinetics, etc. There is. (a) As biological activity or its index, antigen binding activity, in vitro cytotoxic activity, in vivo antitumor activity, etc. can be exemplified. For example, the dissociation constant (KD value) for HLA/NY-ESO is 100 nM or less or 50 nM or less, preferably 20 nM or less or 10 nM or less, more preferably 5 nM or less. Furthermore, for example, the EC 50 value of cytotoxic activity exerted against endogenous human NY-ESO expressing cell lines U266B1 and/or NCI-H1703 using human peripheral blood mononuclear cells as effector cells is 20 nM. Hereinafter, it is preferably 10 nM or less, more preferably 5 nM or less (the method described in Example 8 of WO2021/200857 can be exemplified as an example of measuring and calculating in vitro cytotoxic activity, but is not limited thereto). Furthermore, for example, 0.1 mL of a suspension of 6 × 10 7 cells/mL of human squamous cell lung cancer cell line NCI-H1703 was subcutaneously transplanted into NOG mice, and 4 days later, 3.75 × 10 cells/mL of human peripheral blood mononuclear cells were transplanted subcutaneously into NOG mice. 0.2 mL of 7 cells/mL suspension was implanted into the tail vein, and after 14 days, the antibody was administered three times once a week. Tumor growth inhibitory activity was measured relative to the control group administered with vehicle. is 50% or more, preferably 75% or more, more preferably 90% or more (the method described in Example 9 can be exemplified as an example of measuring and calculating in vivo antitumor activity, but is not limited thereto) . (b) Since impurities contained in biopharmaceuticals affect the safety of pharmaceuticals, it is necessary to appropriately set standards and control whether or not they increase during manufacturing and storage. Among them, HMWS (aggregates) is one of the major impurities and is an item that should be particularly strictly controlled because it is also involved in immunogenicity risk and reduction in drug efficacy. Management of impurities needs to be evaluated not only during manufacturing, but also including stability (presence or absence of increase) over time during the manufacturing process and after manufacturing. The shelf life of a drug is determined based on the results of long-term stability tests, so antibodies that are stable over time can have a longer shelf life. Therefore, the physicochemical properties in the present invention that serve as indicators for selecting suitable antibodies for biopharmaceuticals include acid resistance (HMWS production inhibition, etc.) and solution stability (HMWS production inhibition, etc.). In addition, as another indicator, a host cell suitable for producing the antibody of the present invention, a binding fragment thereof, or a molecule containing them, such as Expi293F cells, may be used to introduce a gene encoding the amino acid sequence contained therein. Examples include high yield and high yield in a culture of recombinant cells. Suitable antibodies of the invention, antigen-binding fragments thereof and multispecific antibodies containing them with their physico-chemical property characteristics: allow their solution to be exposed to acidic conditions and their manufacture; For example, it becomes possible or easy to carry out processes such as protein A, chromatography such as ion exchange, and virus inactivation; even if they are used as a solution, the production of HMWS can be suppressed to a low level, so it is easy to carry out their production, formulation, It becomes possible or easy to distribute and preserve pharmaceuticals containing them; it becomes possible to efficiently produce them. Regarding acid resistance, for example, the content of HMWS calculated by incubating at pH 3.5, room temperature for 1 hour, and then measuring HMWS by size exclusion chromatography is 5% or less, preferably 2% or less, more preferably is 1% or less (the method described in Example 19-1 of WO2021/200857 can be exemplified as an example of measuring and calculating the HMWS content for acid resistance evaluation, but is not limited thereto). Solution stability is calculated by, for example, dissolving the solution in pH 6.0 consisting of 25mM histidine and 5% sorbitol to a concentration of 25mg/ml, storing it at 25°C for 6 days, and then measuring HMWS by size exclusion chromatography. The content of HMWS is 20% or less, preferably 10% or less (for measuring and calculating the HMWS content for solution stability evaluation, the method described in Example 19-2 of WO2021/200857 is exemplified) (but not limited to). Examples of methods for measuring and calculating production efficiency or yield include, but are not limited to, the methods described in Examples 20 and 21 of WO2021/200857. (c) As safety or its indicators, antigen recognition characteristics, findings at the time of administration, etc. can be exemplified. For example, it recognizes multiple amino acids on the wild-type NY-ESO peptide and does not bind to homologous peptides with amino acid sequences similar but not identical to the wild-type NY-ESO peptide, resulting in side effects due to off-target effects. Low risk. Furthermore, it can be predicted that the immunogenicity is low in the ISPRI web-based immunogenicity screening (EpiVax, Inc), and the risk of side effects such as cytokine production caused by anti-antibodies is low. For example, NYF-0023, NYF-0045, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0082, or NYZ-1010 contained in the bispecific antibody of the present invention can be used as a Balb. When administered to /c mice, no problematic findings were observed regarding blood half-life, weight loss, or other significant toxicity findings. In addition, when NYZ-0082 or NYZ-1010 was administered once to cynomolgus monkeys, no problematic findings were observed regarding blood half-life, and no changes were observed in general condition, body weight, food intake, body temperature measurement, and No treatment-related changes in plasma cytokine levels were observed. (d) As the dynamics or its index, blood half-life etc. can be exemplified. For example, when several bispecific antibodies obtained in the present invention were administered to Balb/c mice or cynomolgus monkeys, no problematic findings regarding blood half-life were observed. The antibodies, binding fragments and molecules thereof of the present invention having such excellent biological activity, physicochemical properties, safety, pharmacokinetics, etc. can be suitably included in pharmaceutical compositions. . Preferred antibodies or antigen-binding fragments thereof of the present invention having the antigen-binding activity described in (a) and the properties described in (b) include NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA- 2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2031, NYA-2047, NYA-2061, NYA-2143 and NYA-3061, more preferably NYA-2047 , NYA-2061, NYA-2143 and NYA-3061, but are not limited thereto. Further, preferred multispecific antibodies of the present invention having the properties described in (a) to (d) include NYF-0016, NYF-0019, NYF-0022, NYF-0023, NYF-0027, NYF-0035, NYF-0044, NYF-0045, NYF-0047, NYF-0048, NYF-0058, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088 and NYZ-1010, more preferably, Examples include NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, NYZ-1007, NYZ-1010, and NYZ-1017, but are not limited thereto.
　本発明において、抗体が結合する「部位」、すなわち抗体が認識する「部位」とは、抗体が結合又は認識する抗原上の部分ペプチド又は部分高次構造を意味する。本発明においては、かかる部位のことをエピトープ、抗体の結合部位とも呼ぶ。本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体が結合又は認識するＨＬＡ／ＮＹ－ＥＳＯ上の部位としては、ＨＬＡ／ＮＹ－ＥＳＯペプチド中の複数のアミノ酸、部分高次構造等を例示することができる。 In the present invention, the "site" to which an antibody binds, that is, the "site" recognized by an antibody, refers to a partial peptide or partial conformation on an antigen that the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site. Examples of the site on HLA/NY-ESO that the anti-HLA/NY-ESO antibody of the present invention binds to or recognizes include multiple amino acids, partial conformation structures, etc. in the HLA/NY-ESO peptide.
　本発明の抗体又は結合断片と「同一の部位に結合する抗体又はその結合断片」も本発明に含まれる。ある抗体と「同一の部位に結合する抗体」とは、該抗体が認識する抗原分子上の部位に結合する他の抗体を意味する。第一抗体が結合する抗原分子上の部分ペプチド又は部分立体構造に第二抗体が結合すれば、第一抗体と第二抗体は同一の部位に結合すると判定することができる。本発明の第一抗体が上記（ａ）記載の抗原結合活性を有する場合、ＨＬＡ／ＮＹ－ＥＳＯ上の同一の部位に結合する第二抗体は、同様の活性を有する蓋然性が極めて高く、かかる第二抗体も本発明に包含される。また、ＨＬＡ／ＮＹ－ＥＳＯへの結合において本発明の第一抗体と競合する抗体も、（ａ）記載の抗原結合活性を有していれば、本発明に包含される。そのような本発明のモノクローナル抗体が認識するＨＬＡ／ＮＹ－ＥＳＯ上の部位に結合する抗体、ＨＬＡ／ＮＹ－ＥＳＯへの結合において本発明のモノクローナル抗体と競合する抗体、及びそれらの結合断片は、好適には、（ａ）記載のｉｎ　ｖｉｔｒｏ細胞傷害活性及びｉｎ　ｖｉｖｏ抗腫瘍活性並びに（ｂ）～（ｄ）記載の性質のうち１つ又は２つ以上、より好適にはそれらの３つ以上、最適には全てを有する。 "Antibodies or binding fragments thereof that bind to the same site" as the antibodies or binding fragments of the present invention are also included in the present invention. An "antibody that binds to the same site" as a certain antibody refers to another antibody that binds to the site on the antigen molecule that the antibody recognizes. If the second antibody binds to a partial peptide or partial three-dimensional structure on the antigen molecule to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same site. If the first antibody of the present invention has the antigen-binding activity described in (a) above, the second antibody that binds to the same site on HLA/NY-ESO is highly likely to have the same activity; Dual antibodies are also encompassed by the invention. Furthermore, antibodies that compete with the first antibody of the present invention in binding to HLA/NY-ESO are also encompassed by the present invention, as long as they have the antigen-binding activity described in (a). Such antibodies that bind to the site on HLA/NY-ESO recognized by the monoclonal antibody of the present invention, antibodies that compete with the monoclonal antibody of the present invention in binding to HLA/NY-ESO, and binding fragments thereof, Preferably, one or more of the in vitro cytotoxic activity and in vivo antitumor activity described in (a) and the properties described in (b) to (d), more preferably three or more thereof, Optimal has everything.
　抗体の結合部位は、免疫アッセイ法など当業者に周知の方法により決定することができる。例えば、抗原のアミノ酸配列をＣ末またはＮ末から適宜削ってなる一連のペプチドを作製し、それらに対する抗体の反応性を検討し、大まかな認識部位を決定した後に、さらに短いペプチドを合成してそれらのペプチドへの抗体の反応性を検討することにより、結合部位を決定することができる。または、例えば、抗原もしくは抗原断片ペプチド等のアミノ酸配列のうち、特定の部位や領域を削除もしくは別のアミノ酸配列に置換もしくはアミノ酸配列に変異を導入し、それらのペプチドへの抗体の反応性を検討することにより、結合部位を決定することができる。抗原断片ペプチドは、遺伝子組換、ペプチド合成等の技術を用いて調製することができる。 The binding site of an antibody can be determined by methods well known to those skilled in the art, such as immunoassay methods. For example, a series of peptides are created by appropriately trimming the amino acid sequence of the antigen from the C-terminus or the N-terminus, the reactivity of the antibody against them is examined, the rough recognition site is determined, and then even shorter peptides are synthesized. The binding site can be determined by examining the reactivity of antibodies to those peptides. Alternatively, for example, a specific site or region of the amino acid sequence of an antigen or antigen fragment peptide, etc. may be deleted or substituted with another amino acid sequence, or mutations may be introduced into the amino acid sequence, and the reactivity of antibodies to those peptides may be examined. By doing so, the binding site can be determined. Antigen fragment peptides can be prepared using techniques such as genetic recombination and peptide synthesis.
　抗体が抗原の部分高次構造に結合又は認識している場合、かかる抗体の結合部位は、Ｘ線構造解析を用いて、当該抗体に隣接する抗原上のアミノ酸残基を特定することにより決定することができる。例えば、抗体又はその断片および抗原又はその断片を結合させて結晶化し、構造解析を行うことにより、抗体と相互作用距離を有する抗原上のアミノ酸残基を特定することができる。相互作用距離は８Å以下であり、好適には６Å以下であり、より好適には４Å以下である。そのような抗体との相互作用距離を有するアミノ酸残基は１つまたはそれ以上で抗体の抗原結合部位（エピトープ）を構成し得る。かかるアミノ酸残基が２つ以上の場合、各アミノ酸は一次配列上で互いに隣接していなくてもよい。 When an antibody binds to or recognizes a partial conformation of an antigen, the binding site of such an antibody is determined by identifying amino acid residues on the antigen adjacent to the antibody using X-ray structural analysis. be able to. For example, amino acid residues on the antigen that have an interaction distance with the antibody can be identified by binding and crystallizing an antibody or its fragment and an antigen or its fragment, and performing structural analysis. The interaction distance is 8 Å or less, preferably 6 Å or less, and more preferably 4 Å or less. One or more of the amino acid residues that have such an interaction distance with the antibody can constitute the antigen-binding site (epitope) of the antibody. When there are two or more such amino acid residues, each amino acid does not need to be adjacent to each other in the primary sequence.
　本発明の抗ＨＬＡ／ＮＹ－ＥＳＯ抗体又はその結合断片は、ＨＬＡ／ＮＹ－ＥＳＯのアミノ酸配列中に存在する複数のアミノ酸を特異的に認識する。それら複数のアミノ酸を認識するか、ＨＬＡ／ＮＹ－ＥＳＯへの結合において本発明の抗体又はその結合断片と競合するか、または、それら複数のアミノ酸と相互作用距離を有する抗体又はその結合断片も、本発明に包含される。さらに、そのような抗体又はその結合断片を含む多重特異性抗体も、本発明に包含される。 The anti-HLA/NY-ESO antibody or binding fragment thereof of the present invention specifically recognizes multiple amino acids present in the amino acid sequence of HLA/NY-ESO. An antibody or binding fragment thereof that recognizes the plurality of amino acids, competes with the antibody or binding fragment thereof of the present invention in binding to HLA/NY-ESO, or has an interaction distance with the plurality of amino acids, Included in the present invention. Furthermore, multispecific antibodies comprising such antibodies or binding fragments thereof are also encompassed by the invention.
 
　本発明の多重特的抗体は、免疫チェックポイント阻害剤と組み合わせて使用することができる。
［免疫チェックポイント阻害剤］
　本発明において「免疫チェックポイント阻害剤」とは、免疫抑制系を阻害し、腫瘍免疫を活性化する薬剤を示し、「免疫チェックポイント分子を阻害する化合物」と同義である。
Multispecific antibodies of the invention can be used in combination with immune checkpoint inhibitors.
[Immune checkpoint inhibitor]
In the present invention, the term "immune checkpoint inhibitor" refers to a drug that inhibits the immunosuppressive system and activates tumor immunity, and has the same meaning as a "compound that inhibits immune checkpoint molecules."
　本発明において使用される免疫チェックポイント阻害剤は、特に制限はないが、好適には、抗ＰＤ－１抗体、抗ＰＤ－Ｌ１抗体、抗ＣＴＬＡ－４抗体、及び抗ＴＩＧＩＴ抗体を挙げることができ、より好適には、抗ＰＤ－１抗体、及び抗ＰＤ－Ｌ１抗体を挙げることができる。　本発明において「抗ＰＤ－１抗体」とは、ＰＤ－１（Ｐｒｏｇｒａｍｍｅｄ　ｃｅｌｌ　ｄｅａｔｈ－１；　ＣＤ２７９；　ＰＤＣＤ１）に特異的に結合する抗体を示し、好適には、ＰＤ－１と、その結合相手である、ＰＤ－Ｌ１やＰＤ－Ｌ２との相互作用から生じるシグナル伝達を低減、阻害、及び／又は干渉する作用を有する抗体を示す。本発明において使用される抗ＰＤ－１抗体としては、臨床での有効性と安全性が確認されていれば特に制限はないが、好適には、ニボルマブ（Ｎｉｖｏｌｕｍａｂ）（国際公開第２００６／１２１１６８号等）、及び、ペムブロリズマブ（Ｐｅｍｂｒｏｌｉｚｕｍａｂ）（国際公開第２００８／１５６７１２号等）を例示することができる。また、本発明において使用される抗ＨＬＡ―Ａ２／ＮＹ―ＥＳＯ抗体を含む多重特異性抗体との併用効果を前臨床研究において確認する目的で、市販の研究用抗ＰＤ－１抗体（例：ｃｌｏｎｅ　ＲＭＰ１－１４）等を用いることもできる。　本発明において「抗ＰＤ－Ｌ１抗体」とは、ＰＤ－Ｌ１（Ｐｒｏｇｒａｍｍｅｄ　ｃｅｌｌ　ｄｅａｔｈ　ｌｉｇａｎｄ　１；　ＣＤ２７４；　Ｂ７－Ｈ１）に特異的に結合する抗体を示し、好適には、ＰＤ－Ｌ１と、その結合相手である、ＰＤ－１やＢ７．１（ＣＤ８０）との相互作用から生じるシグナル伝達を低減、阻害、及び／又は干渉する作用を有する抗体を示す。本発明において使用される抗ＰＤ－Ｌ１抗体としては、臨床での有効性と安全性が確認されていれば特に制限はないが、好適には、アテゾリズマブ（Ａｔｅｚｏｌｉｚｕｍａｂ）（国際公開第２０１０／０７７６３４号等）、デュルバルマブ（Ｄｕｒｖａｌｕｍａｂ）（国際公開第２０１１／０６６３８９号等）、及び、アベルマブ（Ａｖｅｌｕｍａｂ）（国際公開第２０１３／０７９１７４号等）を例示することができる。また、本発明において使用される抗ＨＬＡ―Ａ２／ＮＹ―ＥＳＯ抗体を含む多重特異性抗体との併用効果を前臨床研究において確認する目的で、市販の研究用抗ＰＤ－Ｌ１抗体（例：ｃｌｏｎｅ　１０Ｆ．９Ｇ２）等を用いることもできる。本発明において「抗ＣＴＬＡ－４抗体」とは、ＣＴＬＡ－４（Ｃｙｔｏｔｏｘｉｃ　Ｔ－ｌｙｍｐｈｏｃｙｔｅ－ａｓｓｏｃｉａｔｅｄ　ｐｒｏｔｅｉｎ　４；　ＣＤ１５２）に特異的に結合する抗体を示し、好適には、ＣＴＬＡ－４と、その結合相手である、Ｂ７．１（ＣＤ８０）やＢ７．２（ＣＤ８６）との相互作用から生じるシグナル伝達を低減、阻害、及び／又は干渉する作用を有する抗体を示す。本発明において使用される抗ＣＴＬＡ－４抗体としては、臨床での有効性と安全性が確認されていれば特に制限はないが、好適には、イピリムマブ（Ｉｐｉｌｉｍｕｍａｂ）（国際公開第２００１／０１４４２４号等）、トレメリムマブ（Ｔｒｅｍｅｌｉｍｕｍａｂ）（国際公開第２０００／０３７５０４号等）、スパルタリズマブ　（Ｓｐａｒｔａｌｉｚｕｍａｂ）（国際公開第２０１５／１１２９００号等）、セミプリマブ（Ｃｅｍｉｐｌｉｍａｂ）（国際公開第２０１５／１９６０５１号等）を例示することができる。また、本発明において使用される抗ＨＬＡ―Ａ２／ＮＹ―ＥＳＯ抗体を含む多重特異性抗体との併用効果を前臨床研究において確認する目的で、市販の研究用抗ＣＴＬＡ－４抗体（例：ｃｌｏｎｅ　９Ｈ１０）等を用いることもできる。本発明において「抗ＴＩＧＩＴ抗体」とは、ＴＩＧＩＴ（Ｔ　ｃｅｌｌ　ｉｍｍｕｎｏｒｅｃｅｐｔｏｒ　ｗｉｔｈ　Ｉｇ　ａｎｄ　ＩＴＩＭ　ｄｏｍａｉｎｓ）に特異的に結合する抗体を示し、好適には、ＴＩＧＩＴと、その結合相手である、ＣＤ１５５との相互作用から生じるシグナル伝達を低減、阻害、及び／又は干渉する作用を有する抗体を示す。本発明において使用される抗ＴＩＧＩＴ抗体としては、臨床での有効性と安全性が確認されていれば特に制限はないが、好適には、チラゴルマブ（Ｔｉｒａｇｏｌｕｍａｂ）（国際公開第２０１７／０５３７４８号）、ビボストリマブ（Ｖｉｂｏｓｔｏｌｉｍａｂ）（国際公開第２０１６／０２８６５６号）を例示することができる。また、本発明において使用される抗ＨＬＡ―Ａ２／ＮＹ―ＥＳＯ抗体を含む多重特異性抗体との併用効果を前臨床研究において確認する目的で、市販の研究用抗ＴＩＧＩＴ抗体（例：ｃｌｏｎｅ　１Ｂ４）等を用いることもできる。
　本発明において「免疫チェックポイント阻害剤」が抗体である場合、当該抗体の抗原結合断片も「抗体」の範囲に包含される。本発明において、多重特異性抗体と組み合わせて使用される免疫チェックポイント阻害剤は上記いずれかの抗体の抗原結合断片であってもよく、当該抗原結合断片は他の部分を含んでいてもよい。 The immune checkpoint inhibitor used in the present invention is not particularly limited, but preferably includes anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-TIGIT antibody. , more preferably anti-PD-1 antibodies and anti-PD-L1 antibodies. In the present invention, the term "anti-PD-1 antibody" refers to an antibody that specifically binds to PD-1 (Programmed cell death-1; CD279; PDCD1), preferably an antibody that specifically binds to PD-1 and its binding partner. This refers to an antibody that has the effect of reducing, inhibiting, and/or interfering with certain signal transduction resulting from interaction with PD-L1 or PD-L2. The anti-PD-1 antibody used in the present invention is not particularly limited as long as its clinical efficacy and safety have been confirmed, but Nivolumab (International Publication No. 2006/121168) is preferably used. etc.) and pembrolizumab (WO 2008/156712 etc.). In addition, for the purpose of confirming the effect of combination with multispecific antibodies including anti-HLA-A2/NY-ESO antibodies used in the present invention in preclinical studies, commercially available research anti-PD-1 antibodies (e.g. clone RMP1-14) etc. can also be used. In the present invention, the term "anti-PD-L1 antibody" refers to an antibody that specifically binds to PD-L1 (Programmed cell death ligand 1; CD274; B7-H1), and preferably PD-L1 and its binding. This refers to an antibody that has the effect of reducing, inhibiting, and/or interfering with signal transduction resulting from interaction with partner PD-1 or B7.1 (CD80). The anti-PD-L1 antibody used in the present invention is not particularly limited as long as clinical efficacy and safety have been confirmed, but preferably atezolizumab (International Publication No. 2010/077634) etc.), Durvalumab (WO 2011/066389, etc.), and Avelumab (WO 2013/079174, etc.). In addition, for the purpose of confirming the effect of combination with multispecific antibodies including anti-HLA-A2/NY-ESO antibodies used in the present invention in preclinical studies, commercially available research anti-PD-L1 antibodies (e.g. clone 10F.9G2) etc. can also be used. In the present invention, the term "anti-CTLA-4 antibody" refers to an antibody that specifically binds to CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4; CD152), preferably CTLA-4 and its binding partner. This refers to an antibody that has the effect of reducing, inhibiting, and/or interfering with signal transduction resulting from interaction with B7.1 (CD80) and B7.2 (CD86). The anti-CTLA-4 antibody used in the present invention is not particularly limited as long as clinical efficacy and safety have been confirmed, but preferably Ipilimumab (International Publication No. 2001/014424) ), tremelimumab (WO 2000/037504, etc.), spartalizumab (WO 2015/112900, etc.), cemiplimab (WO 2015/196051, etc.) I can give an example. In addition, for the purpose of confirming the effect of combination with multispecific antibodies including anti-HLA-A2/NY-ESO antibodies used in the present invention in preclinical studies, commercially available research anti-CTLA-4 antibodies (e.g., clone 9H10) etc. can also be used. In the present invention, the term "anti-TIGIT antibody" refers to an antibody that specifically binds to TIGIT (T cell immunoreceptor with Ig and ITIM domains), and preferably the interaction between TIGIT and its binding partner, CD155. Indicates an antibody that has the effect of reducing, inhibiting, and/or interfering with signal transduction resulting from. The anti-TIGIT antibody used in the present invention is not particularly limited as long as clinical efficacy and safety are confirmed, but preferably Tiragolumab (International Publication No. 2017/053748), Vibostolimab (International Publication No. 2016/028656) can be exemplified. In addition, for the purpose of confirming the effect of combination with the multispecific antibody including the anti-HLA-A2/NY-ESO antibody used in the present invention in preclinical research, commercially available anti-TIGIT antibody for research use (e.g. clone 1B4) was used. etc. can also be used.
In the present invention, when the "immune checkpoint inhibitor" is an antibody, the antigen-binding fragment of the antibody is also included in the scope of the "antibody". In the present invention, the immune checkpoint inhibitor used in combination with the multispecific antibody may be an antigen-binding fragment of any of the antibodies described above, and the antigen-binding fragment may contain other moieties.
［化学療法剤］
　本発明において「化学療法剤」とは、抗がん、抗腫瘍活性を有する化合物のうち化学的に合成された薬剤を意味する。 [Chemotherapy agent]
In the present invention, the term "chemotherapeutic agent" refers to a chemically synthesized drug among compounds having anticancer or antitumor activity.
　本発明において使用される化学療法剤は、特に限定されないが、好適には、トポイソメラーゼ阻害剤、微小管阻害剤、プラチナ製剤、ＤＮＡ脱メチル化剤、抗がん性抗生物質、及びアルキル化剤でありトポイソメラーゼ阻害剤の好適な例としては、イリノテカン、トポテカン、エトポシドや、薬物複合体としてイリノテカンの活性代謝物であるＳＮ－３８を結合させたサシツズマブ　ゴビテカン等を挙げることができる。微小管阻害剤の好適な例としては、パクリタキセル、ドセタキセル、ビンクリスチン、ビンブラスチン、ビンデシン、エリブリン、ビノレルビン、アルブミン懸濁型パクリタキセル（Ｎａｂパクリタキセル）等を挙げることができる。プラチナ製剤の好適な例としては、オキサリプラチン、カルボプラチン、シスプラチン、ネダプラチン等を挙げることができる。ＤＮＡ脱メチル化剤の好適な例としては、アザシチジン、デシタビン等を挙げることができる。抗がん性抗生物質の好適な例としては、ドキソルビシン、ブレオマイシン、リポソーマルドキソルビシン等を挙げることができる。アルキル化剤の好適な例としては、イホスファミド、シクロホスファミド、ダカルバジン等を挙げることができる。ただし、本発明の多重特異性抗体と組み合わせて使用される化学療法剤はこれらに限定されるものではない。 The chemotherapeutic agents used in the present invention are not particularly limited, but preferably include topoisomerase inhibitors, microtubule inhibitors, platinum agents, DNA demethylating agents, anticancer antibiotics, and alkylating agents. Suitable examples of topoisomerase inhibitors include irinotecan, topotecan, etoposide, and sacituzumab govitecan, which is conjugated with SN-38, an active metabolite of irinotecan, as a drug complex. Suitable examples of microtubule inhibitors include paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, albumin-suspended paclitaxel (Nab-paclitaxel), and the like. Suitable examples of platinum preparations include oxaliplatin, carboplatin, cisplatin, nedaplatin, and the like. Suitable examples of DNA demethylating agents include azacytidine, decitabine, and the like. Suitable examples of anticancer antibiotics include doxorubicin, bleomycin, liposomal doxorubicin, and the like. Suitable examples of alkylating agents include ifosfamide, cyclophosphamide, dacarbazine, and the like. However, the chemotherapeutic agents used in combination with the multispecific antibodies of the present invention are not limited to these.
［医薬］
　本発明は、多重特異性分子と免疫チェックポイント阻害剤又は化学療法剤が組み合わされてなる医薬組成物及び治療方法を提供する。 [Medicine]
The present invention provides pharmaceutical compositions and therapeutic methods comprising a combination of a multispecific molecule and an immune checkpoint inhibitor or chemotherapeutic agent.
　本発明の医薬組成物及び治療方法は、多重特異性分子と、免疫チェックポイント阻害剤又は化学療法剤が、それぞれ別異の製剤に有効成分として含有され、同時に又は異なる時間に投与されることを特徴とするものであってもよいし、多重特異性分子と、免疫チェックポイント阻害剤又は化学療法剤が、単一の製剤に有効成分として含有され、投与されることを特徴とするものであってもよい。また、抗腫瘍免疫を活性化する作用により改善される疾患の治療用に、本発明に係る多重特異性分子が単一の製剤に有効成分として含有され、投与されるものであってもよい。 The pharmaceutical composition and treatment method of the present invention allows the multispecific molecule and the immune checkpoint inhibitor or chemotherapeutic agent to be contained as active ingredients in separate formulations and administered at the same time or at different times. The multispecific molecule and the immune checkpoint inhibitor or chemotherapeutic agent may be administered as active ingredients in a single formulation. It's okay. Furthermore, the multispecific molecule according to the present invention may be contained as an active ingredient in a single preparation and administered for the treatment of diseases that are improved by the action of activating anti-tumor immunity.
　本発明の医薬組成物及び治療方法は、がんの治療のために使用することができ、好適には、肺癌（非小細胞肺癌を含む）、尿路上皮癌、大腸癌（結腸直腸癌と呼ぶこともあり、結腸癌及び直腸癌を含む）、前立腺癌、卵巣癌、膵癌、乳癌、膀胱癌、胃癌（胃腺癌と呼ぶこともある）、胃食道接合部腺癌、胃腸間質腫瘍、子宮頸癌、食道癌、扁平上皮癌、腹膜癌、肝臓癌、肝細胞癌、子宮内膜癌、子宮癌、唾液腺癌、腎臓癌、外陰部癌、甲状腺癌、陰茎癌、白血病、悪性リンパ腫、形質細胞腫、骨髄腫、神経上皮組織性腫瘍、神経鞘性腫瘍、頭頸部癌、皮膚癌、咽頭癌、胆のう癌、胆管癌、中皮腫、ページェット病、及び肉腫からなる群より選択される少なくとも一つの疾患の治療のために使用することができる。 The pharmaceutical composition and treatment method of the present invention can be used for the treatment of cancer, preferably lung cancer (including non-small cell lung cancer), urothelial cancer, colorectal cancer (colorectal cancer), etc. gastric cancer (sometimes called gastric adenocarcinoma), gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, Cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, selected from the group consisting of plasmacytoma, myeloma, neuroepithelial tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, cholangiocarcinoma, mesothelioma, Paget's disease, and sarcoma. can be used for the treatment of at least one disease.
　本発明の医薬組成物及び治療方法は、がんの主要な治療法である薬物療法のための薬剤として選択して使用することができ、その結果として、がん細胞の成長を遅らせ、増殖を抑え、さらにはがん細胞を破壊することができる。これらの作用によって、がん患者において、がんによる症状からの解放や、ＱＯＬの改善を達成でき、がん患者の生命を保って治療効果が達成される。がん細胞の破壊には至らない場合であっても、がん細胞の増殖の抑制やコントロールによってがん患者においてより高いＱＯＬを達成しつつより長期の生存を達成させることができる。このような薬物療法においての薬物単独での使用の他、本発明の医薬組成物及び治療方法は、アジュバント療法において他の療法と組み合わせる薬剤としても使用でき、外科手術や、放射線療法、ホルモン療法等と組み合わせることができる。さらにはネオアジュバント療法における薬物療法の薬剤として使用することもできる。以上のような治療的使用の他、本発明の医薬組成物及び治療方法は、微細な転移がん細胞の増殖を押さえ、さらには破壊するといった予防効果も期待することができる。例えば、転移過程で体液中にあるがん細胞を抑制し破壊する効果や、いずれかの組織に着床した直後の微細ながん細胞に対する抑制、破壊等の効果が期待できる。したがって、特に外科的な癌の除去後においての癌転移の抑制、予防効果が期待できる。本発明の医薬組成物及び治療方法は、患者に対しては全身療法として適用する他、癌組織に局所的に適用して治療効果を期待することができる。本発明の医薬組成物及び治療方法は、哺乳動物に対して好適に使用することができるが、より好適にはヒトに対して使用することができる。本発明の医薬組成物は、1種以上の薬学的に適合性の成分を含む薬学的組成物として投与され得る。本発明の医薬組成物において使用される物質としては、投与量や投与濃度において、この分野において通常使用される製剤添加物その他から適宜選択して適用することができる。例えば、上記薬学的組成物は、代表的には、1種以上の薬学的キャリア（例えば、滅菌した液体）を含む。ここで液体には、例えば、水及び油（石油、動物起源、植物起源、又は合成起源の油）が含まれる。油は、例えば、ラッカセイ油、大豆油、鉱油、ゴマ油等であってよい。水は、上記薬学的組成物が静脈内投与される場合に、より代表的なキャリアである。食塩水溶液、並びにデキストロース水溶液及びグリセロール水溶液もまた、液体キャリアとして、特に、注射用溶液のために使用され得る。適切な薬学的賦形剤は、この分野で公知のものから適宜選択することができる。上記組成物はまた、所望であれば、微量の湿潤剤若しくは乳化剤、又はpH緩衝化剤を含み得る。適切な薬学的キャリアの例は、Ｅ．　Ｗ．　Ｍａｒｔｉｎによる「Ｒｅｍｉｎｇｔｏｎ’ｓ　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｓｃｉｅｎｃｅｓ」に記載される。その処方は、投与の態様に対応する。 The pharmaceutical composition and treatment method of the present invention can be selected and used as a drug for drug therapy, which is the main treatment method for cancer, and as a result, it slows down the growth of cancer cells and inhibits their proliferation. It can suppress and even destroy cancer cells. Through these actions, relief from cancer-related symptoms and improvement of QOL can be achieved in cancer patients, and therapeutic effects can be achieved while preserving the lives of cancer patients. Even if cancer cells are not destroyed, suppression and control of cancer cell proliferation can help cancer patients achieve a higher quality of life and longer survival. In addition to the use of the drug alone in such drug therapy, the pharmaceutical composition and treatment method of the present invention can also be used as a drug in combination with other therapies in adjuvant therapy, such as surgery, radiotherapy, hormone therapy, etc. Can be combined with Furthermore, it can also be used as a drug for drug therapy in neoadjuvant therapy. In addition to the therapeutic uses as described above, the pharmaceutical composition and treatment method of the present invention can also be expected to have preventive effects such as suppressing the proliferation and even destroying microscopic metastatic cancer cells. For example, it can be expected to have the effect of suppressing and destroying cancer cells present in body fluids during the metastasis process, and the effect of suppressing and destroying minute cancer cells immediately after implantation in any tissue. Therefore, it can be expected to suppress and prevent cancer metastasis, especially after surgical removal of cancer. The pharmaceutical composition and treatment method of the present invention can be applied to patients as a systemic therapy, and can also be applied locally to cancer tissues to expect therapeutic effects. The pharmaceutical composition and treatment method of the present invention can be preferably used for mammals, but more preferably for humans. Pharmaceutical compositions of the invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. The substance to be used in the pharmaceutical composition of the present invention can be appropriately selected from pharmaceutical additives and others commonly used in this field, depending on the dosage and concentration. For example, the pharmaceutical compositions typically include one or more pharmaceutical carriers (eg, sterile liquids). Liquids here include, for example, water and oils (oils of petroleum, animal, vegetable or synthetic origin). The oil may be, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Aqueous saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Appropriate pharmaceutical excipients can be appropriately selected from those known in the art. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of suitable pharmaceutical carriers include E. W. Martin, "Remington's Pharmaceutical Sciences". The formulation will depend on the mode of administration.
　種々の送達システムが公知であり、本発明の医薬組成物を投与するために使用され得る。導入経路としては、皮内、筋肉内、腹腔内、静脈内、及び皮下の経路を挙げることができるが、これらに限定されることはない。投与は、例えば、注入又はボーラス注射によるものであり得る。特定の好ましい実施形態において、本発明において使用される多重特異的分子及び免疫チェックポイント阻害剤又は化学療法剤の投与は、注入によるものである。非経口的投与は、好ましい投与経路である。代表的実施形態において、上記医薬組成物は、ヒトへの静脈内投与に適合した薬学的組成物として、常習的手順に従って処方される。代表的には、静脈内投与のための組成物は、滅菌の等張性の水性緩衝液中の溶液である。必要である場合、上記薬学的組成物はまた、可溶化剤及び注射部位での疼痛を和らげるための局所麻酔剤（例えば、リグノカイン）を含み得る。一般に、上記成分は、例えば、活性剤の量を示すアンプル又はサシェ等に密封してシールされた容器中の乾燥凍結乾燥粉末又は無水の濃縮物として、別個に、又は単位剤形中で一緒に混合して、のいずれかで供給される。上記薬学的組成物が注入によって投与される形態の場合、それは、例えば、滅菌の製薬グレードの水又は食塩水を含む注入ボトルで投薬され得る。上記薬学的組成物が注射によって投与される場合、注射用滅菌水又は食塩水のアンプルは、例えば、上記成分が投与前に混合され得るように、提供され得る。 A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention. Routes of introduction can include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, administration of the multispecific molecules and immune checkpoint inhibitors or chemotherapeutic agents used in the invention is by injection. Parenteral administration is the preferred route of administration. In an exemplary embodiment, the pharmaceutical composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical composition can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the above ingredients may be present separately or together in unit dosage form, e.g., as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. Mixed and supplied either. When the pharmaceutical composition is in a form to be administered by injection, it can be dosed, for example, in an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration.
　本発明の医薬組成物及び治療方法は、本発明に係る多重特異性分子及び免疫チェックポイント阻害剤又は化学療法剤化学療法剤以外の癌治療剤を含んでいてもよい。本発明の医薬組成物及び治療方法は、他の癌治療剤と併用して投与することもでき、これによって抗腫瘍効果を増強させることができる。この様な目的で使用される他の癌治療剤は、本発明の医薬組成物と同時に、別々に、或は連続して個体に投与されてもよいし、それぞれの投与間隔を変えて投与されてもよい。この様な癌治療剤として、ペメトレキセド（Pemetrexed）、ソラフェニブ（Sorafenib）、エベロリムス（Everolims）、タネスピマイシン（Tanespimycin）、ベバシズマブ（Bevacizumab））等を挙げることができるが、抗腫瘍活性を有する薬剤であれば限定されることはない。 The pharmaceutical composition and treatment method of the present invention may contain a cancer therapeutic agent other than the multispecific molecule and immune checkpoint inhibitor or chemotherapeutic agent according to the present invention. The pharmaceutical composition and treatment method of the present invention can also be administered in combination with other cancer therapeutic agents, thereby enhancing the antitumor effect. Other cancer therapeutic agents used for this purpose may be administered to an individual simultaneously with the pharmaceutical composition of the present invention, separately, or consecutively, or may be administered at different intervals. It's okay. Examples of such cancer therapeutic agents include Pemetrexed, Sorafenib, Everolims, Tanespimycin, and Bevacizumab, but these drugs have antitumor activity. There are no limitations.
　本発明において使用される分子標的薬は、特に限定されないが、好適には、ＶＥＧＦ阻害剤、ＦＧＦＲ阻害剤、及びマルチキナーゼ阻害剤でありＶＥＧＦ阻害剤の好適な例としては、ベバシズマブ、ラムシルマブ、ザルトラップ、アキシチニブ等を例示することができる。ＦＧＦＲ阻害剤の好適な例としては、ペミガチニブ、フチバチニブ等を例示することができる。マルチキナーゼ阻害剤の好適な例としては、ソラフェニブ、スニチニブ、パゾパニブ、レゴラフェニブ、レンバチニブ等を例示すことができる。ただし、本発明の多重特異性抗体と組み合わせて使用される化学療法剤はこれらに限定されるものではない。　　　 The molecular target drugs used in the present invention are not particularly limited, but are preferably VEGF inhibitors, FGFR inhibitors, and multikinase inhibitors. Preferred examples of VEGF inhibitors include bevacizumab, ramucirumab, Trap, axitinib, etc. can be exemplified. Suitable examples of FGFR inhibitors include pemigatinib, futibatinib, and the like. Suitable examples of multikinase inhibitors include sorafenib, sunitinib, pazopanib, regorafenib, lenvatinib, and the like. However, the chemotherapeutic agents used in combination with the multispecific antibodies of the present invention are not limited to these.　　　
　この様な医薬組成物は、選択された組成と必要な純度を持つ製剤として、凍結乾燥製剤或は液状製剤として製剤化することができる。凍結乾燥製剤として製剤化する際には、この分野において使用される適当な製剤添加物が含まれる製剤であってもよい。また液剤においても同様にして、この分野において使用される各種の製剤添加物を含む液状製剤として製剤化することができる。 Such pharmaceutical compositions can be formulated as lyophilized or liquid formulations with the selected composition and required purity. When formulated as a lyophilized preparation, the preparation may contain suitable additives used in this field. Similarly, liquid preparations can be formulated as liquid preparations containing various formulation additives used in this field.
　医薬組成物の組成及び濃度は投与方法によっても変化するが、本発明の医薬組成物に含まれる多重特異性分子及び免疫チェックポイント阻害剤又は化学療法剤は、抗原に対する親和性、すなわち、抗原に対する解離定数（Kd値）の点において、親和性が高い（Kd値が低い）ほど、少量の投与量であっても薬効を発揮させことができる。したがって、多重特異性分子及び免疫チェックポイント阻害剤又は化学療法剤の投与量は、抗原との親和性の状況に基づいて設定することもできる。本発明の多重特異性分子及び免疫チェックポイント阻害剤又は化学療法剤をヒトに対して投与する際には、例えば、０．０００１ｍｇ～１００ｍｇである。所定の投与量は１～１８０日に１回投与してもよいし、１日当たり２回、３回、４回又はそれ以上の分割投与とし適当な間隔で投与してもよい。 Although the composition and concentration of the pharmaceutical composition will also vary depending on the method of administration, the multispecific molecules and immune checkpoint inhibitors or chemotherapeutic agents contained in the pharmaceutical compositions of the present invention have an affinity for the antigen, i.e. In terms of dissociation constant (Kd value), the higher the affinity (lower the Kd value), the more effective the drug can be exerted even at a small dose. Therefore, the dosage of the multispecific molecule and immune checkpoint inhibitor or chemotherapeutic agent can also be set based on the affinity situation with the antigen. When administering the multispecific molecule and immune checkpoint inhibitor or chemotherapeutic agent of the present invention to humans, the dose is, for example, 0.0001 mg to 100 mg. A predetermined dose may be administered once every 1 to 180 days, or may be divided into two, three, four or more doses per day and administered at appropriate intervals.
　本発明の多重特異性分子の場合、投与方法として、例えば、０．００１ｍｇ／ｋｇ～８ｍｇ/ｋｇを3週に1度投与する方法を挙げることができる。また、投与は3週に1度（ｑ３ｗ）であればよいが、1週に1度（ｑ１ｗ）、2週に1度（ｑ２ｗ）、または4週に1度（ｑ４ｗ）であってもよい。
以下に示す例によって本発明を具体的に説明するが、本発明はこれらに限定されるものではない。また、これらはいかなる意味においても限定的に解釈されるものではない。 In the case of the multispecific molecule of the present invention, the administration method includes, for example, a method in which 0.001 mg/kg to 8 mg/kg is administered once every three weeks. In addition, administration may be done once every three weeks (q3w), but it may also be administered once a week (q1w), once every two weeks (q2w), or once every four weeks (q4w). .
The present invention will be specifically explained with reference to the following examples, but the present invention is not limited thereto. Furthermore, these are not to be construed as limiting in any way.
　以下、実施例において本発明を更に詳細に説明するが、本発明はこれらに限定されない。 Hereinafter, the present invention will be explained in more detail in Examples, but the present invention is not limited thereto.
　なお、下記実施例において遺伝子操作に関する各操作は特に明示がない限り、当業者が使用する実験書に記載の方法により行うか、または、市販の試薬やキットを用いる場合には市販品の指示書に従って行った。 In the examples below, unless otherwise specified, each operation related to genetic manipulation is performed by the method described in the experimental manual used by those skilled in the art, or if commercially available reagents or kits are used, the instructions for the commercially available product are used. I followed.
実施例１．　ヒト扁平上皮肺がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）とＰａｃｌｉｔａｘｅｌとのｉｎ　ｖｉｖｏ併用効果
　ヒト扁平上皮肺がん細胞株ＮＣＩ－Ｈ１７０３（ＡＴＣＣ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで６×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約１週間後（Ｄａｙ６～７）より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 1. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Paclitaxel on human squamous cell lung cancer cell line Human squamous cell lung cancer cell line NCI-H1703 (ATCC ) was prepared at 6×10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 )=average estimated tumor volume of each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ１２に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、ヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５×１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０２ ｍｇ／ｋｇ：ＷＯ２０２１／２００８５７記載の方法により調製した。）を尾静脈内投与した。またＰａｃｌｉｔａｘｅｌ（２０ ｍｇ／ｋｇ）を腹腔内投与した。投与はＤａｙ１３、Ｄａｙ２０に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 12, groups were divided according to tumor volume so that n=5 in each group, and human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells to 5 x 10 7 cells/mL with PBS. 0.2 mL was implanted into the tail vein. After 3-4 hours, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg/kg: prepared by the method described in WO2021/200857) was administered into the tail vein. . Paclitaxel (20 mg/kg) was also administered intraperitoneally. Administration was carried out on Day 13 and Day 20. Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、Ｐａｃｌｉｔａｘｅｌ単独投与、及びＮＹＺ－１０１０とＰａｃｌｉｔａｘｅｌ併用投与の結果を図１（１）に示した。移植３１日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で７９．８％、Ｐａｃｌｉｔａｘｅｌ単独投与群で７４．１％であったのに対し、ＮＹＺ－１０１０とＰａｃｌｉｔａｘｅｌ併用群では９９．１％であり、併用により抗腫瘍効果が増強されることが示された。またＤａｙ１２の腫瘍体積をベースラインとした各個体のＤａｙ３１の腫瘍体積の変化率Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）を以下に示す計算式により算出し、図１（２）に示した。また各群のＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）平均値を表１に記載した。ＮＹＺ－１０１０とＰａｃｌｉｔａｘｅｌの併用による腫瘍退縮効果が示された。 The results of NYZ-1010 alone administration, Paclitaxel alone administration, and NYZ-1010 and Paclitaxel combination administration are shown in Figure 1 (1). TGI 31 days after transplantation was 79.8% in the NYZ-1010 single administration group and 74.1% in the Paclitaxel single administration group, whereas it was 99.1% in the NYZ-1010 and Paclitaxel combination group. It was shown that the antitumor effect was enhanced by combined use. Furthermore, the rate of change from baseline (%) in the tumor volume on Day 31 of each individual using the tumor volume on Day 12 as the baseline was calculated using the formula shown below, and is shown in FIG. 1 (2). In addition, the average value of Change from baseline (%) for each group is listed in Table 1. The tumor regression effect of the combination of NYZ-1010 and Paclitaxel was demonstrated.
　Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）＝各個体の（Ｄａｙ３１におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ－Ｄａｙ１２におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ）／Ｄａｙ１２におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００ Change from baseline (%) = (Estimated Tumor Volume at Day 31 - Estimated Tumor Volume at Day 12) of each individual / Estimated T at Day 12 umor Volume×100
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
実施例２．　ヒト扁平上皮肺がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）とＮａｂ　Ｐａｃｌｉｔａｘｅｌとのｉｎ　ｖｉｖｏ併用効果
　ヒト扁平上皮肺がん細胞株ＮＣＩ－Ｈ１７０３（ＡＴＣＣ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで６×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約１週間後（Ｄａｙ６～７）より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 2. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Nab Paclitaxel on human squamous cell lung cancer cell line Human squamous cell lung cancer cell line NCI-H1703 ( ATCC) was prepared at 6×10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値:　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 ) = Average value of estimated tumor volume of each individual: Estimated tumor volume (mm 3 ) of each individual = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ１３に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、ヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５×１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０２ ｍｇ／ｋｇ）を尾静脈内投与した。またＮａｂ Ｐａｃｌｉｔａｘｅｌ（２０ ｍｇ／ｋｇ）を腹腔内投与した。投与はＤａｙ１３、Ｄａｙ２０に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 13, grouping was performed based on tumor volume so that n = 5 in each group, and human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells to 5 x 10 7 cells/mL with PBS. 0.2 mL was implanted into the tail vein. After 3-4 hours, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg/kg) was administered into the tail vein. In addition, Nab Paclitaxel (20 mg/kg) was administered intraperitoneally. Administration was carried out on Day 13 and Day 20. Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、Ｎａｂ Ｐａｃｌｉｔａｘｅｌ単独投与、及びＮＹＺ－１０１０とＮａｂ Ｐａｃｌｉｔａｘｅｌ併用投与の結果を図２（１）に示した。移植３８日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で５５．４％、Ｎａｂ Ｐａｃｌｉｔａｘｅｌ単独投与群で８６．３％であったのに対し、ＮＹＺ－１０１０とＮａｂ Ｐａｃｌｉｔａｘｅｌ併用群では１００％であり、併用により抗腫瘍効果が増強されることが示された。とりわけＮａｂ Ｐａｃｌｉｔａｘｅｌとの併用では、３８日において５例中５例に腫瘍の完全消失が認められた。またＤａｙ１３の腫瘍体積をベースラインとした各個体のＤａｙ３８の腫瘍体積の変化率Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）を以下に示す計算式により算出し、図１（２）に示した。また各群のＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）平均値を表２に記載した。ＮＹＺ－１０１０とＮａｂ Ｐａｃｌｉｔａｘｅｌの併用による腫瘍退縮効果が示された。 The results of NYZ-1010 administration alone, Nab Paclitaxel administration alone, and NYZ-1010 and Nab Paclitaxel combination administration are shown in Figure 2 (1). TGI 38 days after transplantation was 55.4% in the NYZ-1010 monotherapy group and 86.3% in the Nab Paclitaxel monotherapy group, whereas it was 100% in the NYZ-1010 and Nab Paclitaxel combination group. It was shown that the antitumor effect was enhanced by combined use. In particular, when used in combination with Nab Paclitaxel, complete disappearance of tumors was observed in 5 out of 5 cases on day 38. Furthermore, the rate of change from baseline (%) in the tumor volume on Day 38 for each individual with the tumor volume on Day 13 as the baseline was calculated using the formula shown below, and is shown in FIG. 1 (2). In addition, the average value of Change from baseline (%) for each group is listed in Table 2. The tumor regression effect of the combination of NYZ-1010 and Nab Paclitaxel was demonstrated.
　Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）＝各個体の（Ｄａｙ３８におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ－Ｄａｙ１３におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ）／Ｄａｙ１３におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００ Change from baseline (%) = (Estimated Tumor Volume at Day 38 - Estimated Tumor Volume at Day 13) of each individual / Estimated T at Day 13 umor Volume×100
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
実施例３．　ヒト肺腺がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）と抗ＰＤ－１抗体Ｐｅｍｂｒｏｌｉｚｕｍａｂとのｉｎ　ｖｉｖｏ併用効果
　ヒト肺腺がん細胞株ＮＣＩ－Ｈ５２２（ＡＴＣＣ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで５×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約２週間後より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 3. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-PD-1 antibody Pembrolizumab on human lung adenocarcinoma cell lines Human lung adenocarcinoma Cell line NCI-H522 (ATCC) was prepared at 5 x 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4-6 weeks old). I did it (Day 0). After about 2 weeks, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 )=average estimated tumor volume of each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ２５に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、ヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５×１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０１ ｍｇ／ｋｇ）を尾静脈内投与した。またＰｅｍｂｒｏｌｉｚｕｍａｂ（Ｍｅｒｃｋ社、１０ ｍｇ／ｋｇ）を尾静脈内投与した。投与はＤａｙ２５、Ｄａｙ３２、Ｄａｙ３９（ＮＹＺ－１０１０）、もしくはＤａｙ２８、Ｄａｙ３２、Ｄａｙ３５、Ｄａｙ３９、Ｄａｙ４２（Ｐｅｍｂｒｏｌｉｚｕｍａｂ）に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 25, grouping was performed based on tumor volume so that n = 5 in each group, and human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells to 5 x 10 7 cells/mL with PBS. 0.2 mL was implanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg/kg) was administered into the tail vein. Pembrolizumab (Merck, 10 mg/kg) was also administered into the tail vein. Administration was performed on Day 25, Day 32, Day 39 (NYZ-1010), or Day 28, Day 32, Day 35, Day 39, Day 42 (Pembrolizumab). Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、Ｐｅｍｂｒｏｌｉｚｕｍａｂ単独投与、及びＮＹＺ－１０１０とＰｅｍｂｒｏｌｉｚｕｍａｂ併用投与の結果を図３に示した。移植５３日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で５０．５％、Ｐｅｍｂｒｏｌｉｚｕｍａｂ単独投与群で３．１％であったのに対し、ＮＹＺ－１０１０とＰｅｍｂｒｏｌｉｚｕｍａｂ併用群では７９.０％であり、併用により抗腫瘍効果が増強されることが示された。 The results of NYZ-1010 alone administration, Pembrolizumab alone administration, and NYZ-1010 and Pembrolizumab combination administration are shown in FIG. 3. TGI 53 days after transplantation was 50.5% in the NYZ-1010 monotherapy group and 3.1% in the Pembrolizumab monotherapy group, whereas it was 79.0% in the NYZ-1010 and Pembrolizumab combination group. It was shown that the antitumor effect was enhanced by combined use.
実施例４．　ヒトＣＤ３εノックインマウスの作製
　抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子はヒトＣＤ３εには結合するものの、げっ歯類のＣＤ３εには交差しない。そこで抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子のｉｎ　ｖｉｖｏ での評価を可能にするため、ヒトＣＤ３εノックインマウスを作製した。マウスＣＤ３εにＣＲＩＳＰＲ－Ｃａｓ９と抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子の結合するヒトＣＤ３ε配列をノックインしたベクターを作製し、マイクロインジェクション法にてＣ５７ＢＬ／６Ｊマウスの受精卵に導入した。インジェクション後の受精卵を雌マウスの卵管もしくは子宮に移植し、得られた産仔の尾部組織を生検しＰＣＲおよびシーケンス解析を実施した。目的の変異を持つ個体を選別し、以降の実験に供した。 Example 4. Generation of human CD3ε knock-in mice The anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule binds to human CD3ε but does not cross-react to rodent CD3ε. Therefore, in order to enable in vivo evaluation of the anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule, human CD3ε knock-in mice were generated. A vector was created in which a human CD3ε sequence that binds CRISPR-Cas9 and an anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule was knocked into mouse CD3ε, and the vector was injected into fertilized eggs of C57BL/6J mice using the microinjection method. Introduced. The fertilized eggs after injection were transplanted into the oviduct or uterus of a female mouse, and the tail tissues of the resulting offspring were biopsied and subjected to PCR and sequence analysis. Individuals with the desired mutation were selected and subjected to subsequent experiments.
実施例５．　ヒトＣＤ３εノックインマウスを用いたＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＦ－００１６）と抗マウスＰＤ－１抗体のｉｎ　ｖｉｖｏ併用効果
　Ｎａｔｉｏｎａｌ　Ｃａｎｃｅｒ　Ｉｎｓｔｉｔｕｔｅ社より導入したマウス大腸がん細胞株ＭＣ－３８にレトロウイルスベクターを用いてＨＬＡ－Ａ２、ＮＹ－ＥＳＯ、β２Ｍ　Ｓｉｎｇｌｅ　Ｃｈａｉｎ　Ｔｒｉｍｅｒ（ＮＹ－ＳＣＴと略記)を導入したＭＣ－３８　ＮＹ－ＳＣＴ細胞を用いた。この細胞は細胞膜上にＨＬＡ－Ａ２／ＮＹ－ＥＳＯを発現している。ＭＣ－３８　ＮＹ－ＳＣＴをＰＢＳ（Ｗａｋｏ社）で８×１０６細胞／ｍＬになるよう調製し、実施例４で作製したヒトＣＤ３εノックインマウス（雌性、６～７週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。Ｄａｙ７より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 5. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYF-0016) and anti-mouse PD-1 antibody using human CD3ε knock-in mice Introduced from National Cancer Institute MC-38 NY-SCT cells were used, in which HLA-A2, NY-ESO, and β2M Single Chain Trimer (abbreviated as NY-SCT) were introduced into mouse colon cancer cell line MC-38 using a retrovirus vector. This cell expresses HLA-A2/NY-ESO on the cell membrane. MC-38 NY-SCT was prepared at 8×10 6 cells/mL in PBS (Wako) and injected subcutaneously into the human CD3ε knock-in mouse (female, 6-7 weeks old) prepared in Example 4 at 0. 1 mL was transplanted (Day 0). From Day 7, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 ) = Average value of estimated tumor volume of each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ７に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＦ－００１６、５ ｍｇ／ｋｇ：ＷＯ２０２１／２００８５７記載の方法により調製した。）を尾静脈内投与した。また抗マウスＰＤ－１抗体 (ｃｌｏｎｅ　ＲＭＰ１－１４、Ｂｉｏ　Ｘ　Ｃｅｌｌ社、５ ｍｇ／ｋｇ)を尾静脈内投与した。投与はＤａｙ７、Ｄａｙ１０に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 7, groups were divided by tumor volume so that n = 5 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYF-0016, 5 mg/kg: described in WO2021/200857) was administered. ) was administered intravenously into the tail vein. In addition, an anti-mouse PD-1 antibody (clone RMP1-14, BioX Cell, 5 mg/kg) was administered into the tail vein. Administration was carried out on Day 7 and Day 10. Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＦ－００１６単独投与、抗マウスＰＤ－１抗体単独投与、及びＮＹＦ－００１６と抗マウスＰＤ－１抗体併用投与の結果を図４に示した。移植１７日後におけるＴＧＩは、ＮＹＦ－００１６単独投与群で３１．５％、抗マウスＰＤ－１抗体単独投与群で３５．３％であったのに対し、ＮＹＦ－００１６と抗マウスＰＤ－１抗体併用群では５７．５％であり、併用により抗腫瘍効果が増強されることが示された。 The results of administration of NYF-0016 alone, anti-mouse PD-1 antibody alone, and combined administration of NYF-0016 and anti-mouse PD-1 antibody are shown in FIG. TGI 17 days after transplantation was 31.5% in the NYF-0016 monotherapy group and 35.3% in the anti-mouse PD-1 antibody monotherapy group, whereas NYF-0016 and anti-mouse PD-1 antibody therapy In the combination group, it was 57.5%, indicating that the antitumor effect was enhanced by the combination.
実施例６．　ヒト肺腺がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）と抗ＰＤ－１抗体Ｐｅｍｂｒｏｌｉｚｕｍａｂ及びマルチキナーゼ阻害剤ｌｅｎｖａｔｉｎｉｂとのｉｎ　ｖｉｖｏ併用効果
　ヒト肺腺がん細胞株ＮＣＩ－Ｈ５２２（ＡＴＣＣ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで５×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約３週間後より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 6. In vivo combination of Fc-attached anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) with anti-PD-1 antibody Pembrolizumab and multikinase inhibitor lenvatinib against human lung adenocarcinoma cell lines Effect Human lung adenocarcinoma cell line NCI-H522 (ATCC) was prepared at 5 x 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and cultured in NSG mice (female, 4-6 weeks old). 0.1 mL was subcutaneously transplanted (Day 0). After about 3 weeks, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 ) = Average value of estimated tumor volume of each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ２８に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、ヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５×１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０１ ｍｇ／ｋｇ）を尾静脈内投与した。またＰｅｍｂｒｏｌｉｚｕｍａｂ（Ｍｅｒｃｋ社、１０ ｍｇ／ｋｇ）を腹腔内投与、ｌｅｎｖａｔｉｎｉｂ（３０ｍｐｋ）を経口投与した。投与はＤａｙ２８、Ｄａｙ３５、Ｄａｙ４２（ＮＹＺ－１０１０）、Ｄａｙ３２、Ｄａｙ３９、Ｄａｙ４６（Ｐｅｍｂｒｏｌｉｚｕｍａｂ）、もしくはＤａｙ２８からＤａｙ３２まで一日一回、Ｄａｙ３５からＤａｙ３９まで一日一回、Ｄａｙ４２からＤａｙ３６まで一日一回（ｌｅｎｖａｔｉｎｉｂ）に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 28, groups were divided according to tumor volume so that n=5 in each group, and human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells to 5 x 10 7 cells/mL with PBS. 0.2 mL was implanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg/kg) was administered into the tail vein. In addition, Pembrolizumab (Merck, 10 mg/kg) was administered intraperitoneally, and lenvatinib (30 mpk) was administered orally. Administration is on Day 28, Day 35, Day 42 (NYZ-1010), Day 32, Day 39, Day 46 (Pembrolizumab), or once a day from Day 28 to Day 32, once a day from Day 35 to Day 39, once a day from Day 42 to Day 36 ( lenvatinib). Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、ｌｅｎｖａｔｉｎｉｂとＰｅｍｂｒｏｌｉｚｕｍａｂ併用投与、及びＮＹＺ－１０１０とｌｅｎｖａｔｉｎｉｂとＰｅｍｂｒｏｌｉｚｕｍａｂ併用投与の結果を図７２（１）に示した。移植５２日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で５１．４％、ｌｅｎｖａｔｉｎｉｂとＰｅｍｂｒｏｌｉｚｕｍａｂ併用投与群で５０．６％であったのに対し、ＮＹＺ－１０１０とＬｅｎｖａｔｉｎｉｂとＰｅｍｂｒｏｌｉｚｕｍａｂ併用群では８４．１％となり、３剤併用により抗腫瘍効果が増強されることが示された。また、Ｄａｙ２８の腫瘍体積をベースラインとした各個体のＤａｙ５２の腫瘍体積の変化率　Ｃｈａｎｇｅ　ｆｒоｍ　ｂａｓｅｌｉｎｅ（％）を以下に示す計算式により算出し、図７２（２）に示した。各群のＣｈａｎｇｅ　ｆｒоｍ　ｂａｓｅｌｉｎｅ（％）平均値を表３に記載した。ＮＹＺ－１０１０とｌｅｎｖａｔｉｎｉｂ、Ｐｅｍｂｒｏｌｉｚｕｍａｂの併用による腫瘍退縮効果が示された。 The results of the single administration of NYZ-1010, the combined administration of lenvatinib and Pembrolizumab, and the combined administration of NYZ-1010, lenvatinib, and Pembrolizumab are shown in FIG. 72 (1). TGI 52 days after transplantation was 51.4% in the NYZ-1010 alone group and 50.6% in the lenvatinib and Pembrolizumab combination group, whereas it was 84.1 in the NYZ-1010, Lenvatinib, and Pembrolizumab combination group. %, indicating that the antitumor effect was enhanced by the combination of the three drugs. In addition, the rate of change in tumor volume on Day 52 of each individual (Change from baseline (%)) using the tumor volume on Day 28 as the baseline was calculated using the formula shown below, and is shown in FIG. 72 (2). The average Change from baseline (%) values for each group are listed in Table 3. A tumor regression effect was demonstrated by the combination of NYZ-1010, lenvatinib, and pembrolizumab.
　　Ｃｈａｎｇｅ　ｆｒоｍ　ｂａｓｅｌｉｎｅ（％） ＝各個体の（Ｄａｙ５２におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ－Ｄａｙ２８におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ）／Ｄａｙ２８におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００  Change from baseline (%) = (Estimated Tumor Volume at Day 52 - Estimated Tumor Volume at Day 28) of each individual / Estimated at Day 28 Tumor Volume×100
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
実施例７．　ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスの作製
実施例４で作製したＣ５７ＢＬ／６Ｊマウス系統のヒトＣＤ３εノックインマウスをＢＡＬＢ／Ｃ系統と戻し交配し、ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスを作製した。得られた産仔の尾部組織をＰＣＲおよびシークエンス解析を実施した。ホモ置換率の高い個体を選別して繁殖し、以降の実験に供した。 Example 7. Preparation of BALB/C strain human CD3ε knock-in mouse The C57BL/6J mouse strain human CD3ε knock-in mouse produced in Example 4 was backcrossed with the BALB/C strain to generate a BALB/C strain human CD3ε knock-in mouse. PCR and sequence analysis were performed on the tail tissues of the resulting offspring. Individuals with a high homosubstitution rate were selected and bred, and used for subsequent experiments.
 実施例８．　ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスを用いたＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）と抗マウスＰＤ－１抗体のｉｎ　ｖｉｖｏ併用効果
　ＡＴＣＣ社より導入したマウス大腸がん細胞株ＣＴ２６．ＷＴにレトロウイルスベクターを用いてＨＬＡ－Ａ２、ＮＹ－ＥＳＯ、β２Ｍ　Ｓｉｎｇｌｅ　Ｃｈａｉｎ　Ｔｒｉｍｅｒ（ＮＹ－ＳＣＴと略記)を導入したＣＴ２６　ＮＹ－ＳＣＴ細胞を用いた。この細胞は細胞膜上にＨＬＡ－Ａ２／ＮＹ－ＥＳＯを発現している。ＣＴ２６　ＮＹ－ＳＣＴをＰＢＳ（Ｗａｋｏ社）で１×１０７細胞／ｍＬになるよう調製し、実施例７で作製したＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウス（雌性、７～９週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ０）。Ｄａｙ８より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 8. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse PD-1 antibody using BALB/C strain human CD3ε knock-in mice From ATCC The introduced mouse colon cancer cell line CT26. CT26 NY-SCT cells, in which HLA-A2, NY-ESO, and β2M Single Chain Trimer (abbreviated as NY-SCT) were introduced into WT using a retrovirus vector, were used. This cell expresses HLA-A2/NY-ESO on the cell membrane. CT26 NY-SCT was prepared with PBS (Wako) at 1 x 10 7 cells/mL, and injected subcutaneously into the BALB/C strain human CD3ε knock-in mouse (female, 7 to 9 weeks old) prepared in Example 7. 0.1 mL was transplanted (Day 0). From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
 　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 )=average estimated tumor volume of each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ８に各群ｎ＝５～１０になるよう腫瘍体積による群分けを実施し、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、５ ｍｇ／ｋｇ）を尾静脈内投与した。また抗マウスＰＤ－１抗体 (ｃｌｏｎｅ　ＲＭＰ１－１４、Ｂｉｏ　Ｘ　Ｃｅｌｌ社、５ ｍｇ／ｋｇ)を腹腔内投与した。投与はＤａｙ８に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 8, groups were divided according to tumor volume so that n = 5 to 10 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg/kg) was injected into the tail vein. Administered intravenously. In addition, anti-mouse PD-1 antibody (clone RMP1-14, BioX Cell, 5 mg/kg) was administered intraperitoneally. Administration was carried out on Day 8. Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、抗マウスＰＤ－１抗体単独投与、及びＮＹＺ－１０１０と抗マウスＰＤ－１抗体併用投与の結果を図７３（１）に示した。移植２１日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で７０．８％、抗マウスＰＤ－１抗体単独投与群で３８．０％であったのに対し、ＮＹＺ－１０１０と抗マウスＰＤ－１抗体併用群では８４．８％であり、併用により抗腫瘍効果が増強されることが示された。 The results of administration of NYZ-1010 alone, anti-mouse PD-1 antibody alone, and combined administration of NYZ-1010 and anti-mouse PD-1 antibody are shown in FIG. 73 (1). TGI 21 days after transplantation was 70.8% in the NYZ-1010 alone administration group and 38.0% in the anti-mouse PD-1 antibody alone administration group, whereas NYZ-1010 and anti-mouse PD-1 antibody In the combination group, it was 84.8%, indicating that the antitumor effect was enhanced by the combination.
　図７３（２）。各単独投与群では腫瘍が残存し、薬効が限定的であった（それぞれ灰色の実線と細かい破線）のに対し、ＮＹＺ－１０１０と抗マウスＰＤ－１抗体併用群１０例中３例の推定腫瘍体積は０ｍｍ３を示し、その後も完全退縮を維持した（黒丸、実線）。ＣＴ２６　ＮＹ－ＳＣＴを完全退縮マウスに再移植、あるいは腫瘍移植経験のないＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウス（以下、Ｎａiｖｅマウスと表記、雌性、７～９週齢）に移植した。ＣＴ２６　ＮＹ－ＳＣＴはＰＢＳ（Ｗａｋｏ社）で１×１０７細胞／ｍＬになるよう調製し、皮下に０．１ｍＬ移植した（Ｄａｙ５９）。Ｎａiｖｅマウスでは腫瘍が生着して増殖したが（粗い破線）、完全退縮マウスでは腫瘍が生着せず、ＮＹＺ－１０１０と抗マウスＰＤ－１抗体による完全退縮が維持され（黒丸、実線）、腫瘍細胞に対する免疫記憶が成立したことが示された。
実施例９．　ヒト肺腺がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）と抗ＰＤ－Ｌ１抗体Ｄｕｒｖａｌｕｍａｂとのｉｎ　ｖｉｖｏ併用効果
　ヒト肺腺がん細胞株ＮＣＩ－Ｈ５２２（ＡＴＣＣ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで５×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約１週間後（Ｄａｙ６～７）より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Figure 73(2). Tumors remained in each single administration group, and the drug efficacy was limited (gray solid line and thin broken line, respectively), whereas presumed tumors remained in 3 out of 10 patients in the NYZ-1010 and anti-mouse PD-1 antibody combination group. The volume showed 0 mm3 , and complete regression was maintained thereafter (black circle, solid line). CT26 NY-SCT was retransplanted into completely regressed mice or into BALB/C strain human CD3ε knock-in mice (hereinafter referred to as Naive mice, female, 7 to 9 weeks old) that had no experience of tumor transplantation. CT26 NY-SCT was prepared at 1×10 7 cells/mL with PBS (Wako), and 0.1 mL was implanted subcutaneously (Day 59). In naive mice, the tumor engrafted and grew (rough dashed line), but in mice with complete regression, the tumor did not engraft, and complete regression was maintained by NYZ-1010 and anti-mouse PD-1 antibody (black circles, solid line), and the tumor It was shown that immune memory for the cells was established.
Example 9. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-PD-L1 antibody Durvalumab on human lung adenocarcinoma cell lines Human lung adenocarcinoma Cell line NCI-H522 (ATCC) was prepared at 5 x 10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4-6 weeks old). I did it (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］  Estimated Tumor Volume (mm3) = Average value of estimated tumor volume for each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ２４に各群ｎ＝５になるよう腫瘍体積による群分けを実施した。Ｄａｙ２５にヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５ｘ１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０２ ｍｇ／ｋｇ）を尾静脈内投与した。またＤｕｒｖａｌｕｍａｂ（ＡｓｔｒａＺｅｎｅｃａ社、１０ ｍｇ／ｋｇ）は腹腔内投与した。投与はＤａｙ２５、Ｄａｙ３２（ＮＹＺ－１０１０）、もしくはＤａｙ２８、Ｄａｙ３２、Ｄａｙ３５、Ｄａｙ３９（Ｄｕｒｖａｌｕｍａｂ）に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）、及びＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）は以下に示す計算式により算出した。 On Day 24, the mice were divided into groups based on tumor volume so that n=5 in each group. On Day 25, human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells, which were prepared in PBS to a concentration of 5 x 10 7 cells/mL, and 0.2 mL was transplanted into the tail vein. After 3-4 hours, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg/kg) was administered into the tail vein. Additionally, Durvalumab (AstraZeneca, 10 mg/kg) was administered intraperitoneally. Administration was performed on Day 25, Day 32 (NYZ-1010), or Day 28, Day 32, Day 35, Day 39 (Durvalumab). Tumor Growth Inhibition (%) and Change from baseline (%) were calculated using the formulas shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、Ｄｕｒｖａｌｕｍａｂ単独投与、及びＮＹＺ－１０１０とＤｕｒｖａｌｕｍａｂ併用投与の結果を図７４（１）に示した。移植４９日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で６７．７％、Ｄｕｒｖａｌｕｍａｂ単独投与群で－０．８％であったのに対し、ＮＹＺ－１０１０とＤｕｒｖａｌｕｍａｂ併用群では９２．５％であり、併用により抗腫瘍効果が増強されることが示された。またＤａｙ２４の腫瘍体積をベースラインとした各個体のＤａｙ４９の腫瘍体積の変化率Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）を以下に示す計算式により算出し、図７４（２）に示した。また各群のＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）平均値を表４に記載した。ＮＹＺ－１０１０とＤｕｒｖａｌｕｍａｂの併用による強い腫瘍退縮効果が示された。 The results of NYZ-1010 single administration, Durvalumab single administration, and NYZ-1010 and Durvalumab combination administration are shown in Figure 74 (1). TGI 49 days after transplantation was 67.7% in the NYZ-1010 monotherapy group and -0.8% in the Durvalumab monotherapy group, whereas it was 92.5% in the NYZ-1010 and Durvalumab combination group. , it was shown that the antitumor effect was enhanced by combination use. Furthermore, the rate of change from baseline (%) in the tumor volume on Day 49 for each individual with the tumor volume on Day 24 as the baseline was calculated using the formula shown below, and is shown in FIG. 74 (2). Furthermore, the average value of Change from baseline (%) for each group is listed in Table 4. A strong tumor regression effect was demonstrated by the combination of NYZ-1010 and Durvalumab.
　Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）＝各個体の（Ｄａｙ４９におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ－Ｄａｙ２４におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ）／Ｄａｙ２４におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００ Change from baseline (%) = (Estimated Tumor Volume at Day 49 - Estimated Tumor Volume at Day 24) of each individual / Estimated T at Day 24 umor Volume×100
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
実施例１０．　ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスを用いたＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）と抗マウスＰＤ－Ｌ１抗体のｉｎ　ｖｉｖｏ併用効果
　実施例８で作製したＣＴ２６　ＮＹ－ＳＣＴをＰＢＳ（Ｗａｋｏ社）で１×１０７細胞／ｍＬになるよう調製し、実施例７で作製したＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウス（雌性、６～週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ０）。Ｄａｙ８より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 10. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse PD-L1 antibody using BALB/C strain human CD3ε knock-in mice Example 8 The CT26 NY-SCT prepared in Example 7 was adjusted to 1×10 7 cells/mL with PBS (Wako), and used in the BALB/C strain human CD3ε knock-in mouse (female, 6-weeks old) prepared in Example 7. 0.1 mL was subcutaneously transplanted (Day 0). From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
 　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 )=average estimated tumor volume of each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ８に各群ｎ＝５～１０になるよう腫瘍体積による群分けを実施し、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、５ ｍｇ／ｋｇ）を尾静脈内投与した。また抗マウスＰＤ－Ｌ１抗体 (ｃｌｏｎｅ　１０Ｆ．９Ｇ２、Ｂｉｏ　Ｘ　Ｃｅｌｌ社、５ ｍｇ／ｋｇ)を腹腔内投与した。投与はＤａｙ８に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 8, groups were divided according to tumor volume so that n = 5 to 10 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg/kg) was injected into the tail vein. Administered intravenously. In addition, anti-mouse PD-L1 antibody (clone 10F.9G2, BioX Cell, 5 mg/kg) was administered intraperitoneally. Administration was carried out on Day 8. Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、抗マウスＰＤ－Ｌ１抗体単独投与、及びＮＹＺ－１０１０と抗マウスＰＤ－Ｌ１抗体併用投与の結果を図７５（１）に示した。移植２１日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で６８．７％、抗マウスＰＤ－Ｌ１抗体単独投与群で３６．４％であったのに対し、ＮＹＺ－１０１０と抗マウスＰＤ－Ｌ１抗体併用群では７７．４％であり、併用により抗腫瘍効果が増強されることが示された。 The results of administration of NYZ-1010 alone, anti-mouse PD-L1 antibody alone, and combined administration of NYZ-1010 and anti-mouse PD-L1 antibody are shown in FIG. 75 (1). TGI 21 days after transplantation was 68.7% in the NYZ-1010 alone administration group and 36.4% in the anti-mouse PD-L1 antibody alone administration group, whereas NYZ-1010 and anti-mouse PD-L1 antibody In the combination group, it was 77.4%, indicating that the antitumor effect was enhanced by the combination.
　図７５（２）。各単独投与群では腫瘍が残存し、薬効が限定的であった（それぞれ灰色の実線と細かい破線）のに対し、ＮＹＺ－１０１０と抗マウスＰＤ－Ｌ１抗体併用群１０例中２例の推定腫瘍体積は０ｍｍ３を示し、その後も完全退縮を維持した（黒丸、実線）。レトロウイルスベクターのみを導入したＣＴ２６．ＷＴ（以下、ＣＴ２６　Ｍｏｃｋと表記）を完全退縮マウスに再移植、及びＮａiｖｅマウス（雌性、１５～１７週齢）に移植した。ＣＴ２６　ＭｏｃｋはＰＢＳ（Ｗａｋｏ社）で１×１０７細胞／ｍＬになるよう調製し、皮下に０．１ｍＬ移植した（Ｄａｙ６４）。Ｎａiｖｅマウスでは腫瘍が生着して増殖したが（粗い破線）、完全退縮マウスでは腫瘍が生着せず、ＮＹＺ－１０１０と抗マウスＰＤ－Ｌ１抗体による完全退縮が維持され（黒丸、実線）、腫瘍細胞に対する免疫記憶が成立したことが示された。 Figure 75 (2). Tumors remained in each monoadministration group, and the drug efficacy was limited (solid gray line and fine dashed line, respectively), whereas estimated tumors in 2 out of 10 patients in the NYZ-1010 and anti-mouse PD-L1 antibody combination group remained. The volume showed 0 mm3 , and complete regression was maintained thereafter (black circle, solid line). CT26 into which only the retrovirus vector was introduced. WT (hereinafter referred to as CT26 Mock) was retransplanted into completely regressed mice and into naive mice (female, 15 to 17 weeks old). CT26 Mock was prepared with PBS (Wako) at 1×10 7 cells/mL, and 0.1 mL was implanted subcutaneously (Day 64). In naive mice, the tumor engrafted and grew (rough dashed line), but in mice with complete regression, the tumor did not engraft, and complete regression was maintained by NYZ-1010 and anti-mouse PD-L1 antibody (black circle, solid line), and the tumor It was shown that immune memory for the cells was established.
実施例１１．　ＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウスを用いたＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）と抗マウスＣＴＬＡ－４抗体のｉｎ　ｖｉｖｏ併用効果
　実施例８で作製したＣＴ２６　ＮＹ－ＳＣＴをＰＢＳ（Ｗａｋｏ社）で１×１０７細胞／ｍＬになるよう調製し、実施例７で作製したＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウス（雌性、７～９週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ０）。Ｄａｙ８より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 11. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse CTLA-4 antibody using BALB/C strain human CD3ε knock-in mice Example 8 The CT26 NY-SCT prepared in Example 7 was prepared at 1×10 7 cells/mL with PBS (Wako), and the BALB/C strain human CD3ε knock-in mouse (female, 7 to 9 weeks old) prepared in Example 7 was prepared. 0.1 mL was transplanted subcutaneously (Day 0). From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 ) = Average value of estimated tumor volume for each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ８に各群ｎ＝５～１０になるよう腫瘍体積による群分けを実施し、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、５ ｍｇ／ｋｇ）を尾静脈内投与した。また抗マウスＣＴＬＡ－４抗体 (ｃｌｏｎｅ　９Ｄ９、Ｂｉｏ　Ｘ　Ｃｅｌｌ社、５ ｍｇ／ｋｇ)を腹腔内投与した。投与はＤａｙ８に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 8, groups were divided according to tumor volume so that n = 5 to 10 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg/kg) was injected into the tail vein. Administered intravenously. In addition, anti-mouse CTLA-4 antibody (clone 9D9, BioX Cell, 5 mg/kg) was administered intraperitoneally. Administration was carried out on Day 8. Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００）
 
　ＮＹＺ－１０１０単独投与、抗マウスＣＴＬＡ－４抗体単独投与、及びＮＹＺ－１０１０と抗マウスＣＴＬＡ－４抗体併用投与の結果を図７６（１）に示した。移植２１日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で７０．８％、抗マウスＣＴＬＡ－４抗体単独投与群で１２．１％であったのに対し、ＮＹＺ－１０１０と抗マウスＣＴＬＡ－４抗体併用群では８０．４％であり、併用により抗腫瘍効果が増強されることが示された。 Tumor Growth Inhibition (%) = 100 - (Estimated Tumor Volume of each group/Estimated Tumor Volume of Vehicle Control x 100)

The results of administration of NYZ-1010 alone, anti-mouse CTLA-4 antibody alone, and combined administration of NYZ-1010 and anti-mouse CTLA-4 antibody are shown in FIG. 76 (1). TGI 21 days after transplantation was 70.8% in the NYZ-1010 alone administration group and 12.1% in the anti-mouse CTLA-4 antibody alone administration group, whereas NYZ-1010 and anti-mouse CTLA-4 antibody In the combination group, it was 80.4%, indicating that the antitumor effect was enhanced by the combination.
　図７６（２）。各単独投与群では腫瘍が残存し、薬効が限定的であった（それぞれ灰色の実線と細かい破線）のに対し、ＮＹＺ－１０１０と抗マウスＣＴＬＡ－４抗体併用群１０例中１例の推定腫瘍体積は０ｍｍ３を示し、その後も完全退縮を維持した（黒丸、実線）。ＣＴ２６　ＮＹ－ＳＣＴを完全退縮マウスに再移植、及びＮａiｖｅマウス（雌性、７～９週齢）に移植した。ＣＴ２６　ＮＹ－ＳＣＴはＰＢＳ（Ｗａｋｏ社）で１×１０７細胞／ｍＬになるよう調製し、皮下に０．１ｍＬ移植した（Ｄａｙ５９）。Ｎａiｖｅマウスでは腫瘍が生着して増殖したが（粗い破線）、完全退縮マウスでは腫瘍が生着せず、ＮＹＺ－１０１０と抗マウスＣＴＬＡ－４抗体による完全退縮が維持され（黒丸、実線）、腫瘍細胞に対する免疫記憶が成立したことが示された。 Figure 76(2). Tumors remained in each single administration group, and the drug efficacy was limited (gray solid line and thin broken line, respectively), whereas estimated tumors in 1 out of 10 patients in the NYZ-1010 and anti-mouse CTLA-4 antibody combination group remained. The volume showed 0 mm3 , and complete regression was maintained thereafter (black circle, solid line). CT26 NY-SCT was reimplanted into completely regressed mice and into naive mice (female, 7-9 weeks old). CT26 NY-SCT was prepared at 1×10 7 cells/mL with PBS (Wako), and 0.1 mL was implanted subcutaneously (Day 59). In naive mice, the tumor engrafted and grew (rough dashed line), but in mice with complete regression, the tumor did not engraft, and complete regression was maintained by NYZ-1010 and anti-mouse CTLA-4 antibody (black circles, solid line), and the tumor It was shown that immune memory for the cells was established.
実施例１２．　ヒトＣＤ３εノックインマウスを用いたＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）と抗マウスＴＩＧＩＴ抗体のｉｎ　ｖｉｖｏ併用効果
　実施例８で作製したＣＴ２６　ＮＹ－ＳＣＴをＰＢＳ（Ｗａｋｏ社）で１×１０７細胞／ｍＬになるよう調製し、実施例７で作製したＢＡＬＢ／Ｃ系統ヒトＣＤ３εノックインマウス（雌性、７～９週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ０）。Ｄａｙ８より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 12. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse TIGIT antibody using human CD3ε knock-in mouse CT26 NY- produced in Example 8 SCT was prepared at 1×10 7 cells/mL with PBS (Wako), and 0.1 mL was subcutaneously administered to the BALB/C strain human CD3ε knock-in mouse (female, 7 to 9 weeks old) prepared in Example 7. It was transplanted (Day 0). From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm 3 ) = Average value of estimated tumor volume for each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ８に各群ｎ＝５～１０になるよう腫瘍体積による群分けを実施し、抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、５ ｍｇ／ｋｇ）を尾静脈内投与した。また抗マウスＴＩＧＩＴ抗体 (ｃｌｏｎｅ　１Ｂ４、Ａｂｓｏｌｕｔｅ　Ａｎｔｉｂｏｄｙ社、５ ｍｇ／ｋｇ)を腹腔内投与した。投与はＤａｙ８に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）は以下に示す計算式により算出した。 On Day 8, groups were divided according to tumor volume so that n = 5 to 10 in each group, and anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg/kg) was injected into the tail vein. Administered intravenously. In addition, an anti-mouse TIGIT antibody (clone 1B4, Absolute Antibody, 5 mg/kg) was administered intraperitoneally. Administration was carried out on Day 8. Tumor Growth Inhibition (%) was calculated using the formula shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、抗マウスＴＩＧＩＴ抗体単独投与、及びＮＹＺ－１０１０と抗マウスＴＩＧＩＴ抗体併用投与の結果を図７７（１）に示した。移植２４日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で５５．５％、抗マウスＴＩＧＩＴ抗体単独投与群で－１．２％であったのに対し、ＮＹＺ－１０１０と抗マウスＴＩＧＩＴ抗体併用群では７８．３％であり、併用により抗腫瘍効果が増強されることが示された。 The results of the administration of NYZ-1010 alone, the anti-mouse TIGIT antibody alone, and the combined administration of NYZ-1010 and anti-mouse TIGIT antibody are shown in FIG. 77(1). TGI 24 days after transplantation was 55.5% in the NYZ-1010 alone administration group and -1.2% in the anti-mouse TIGIT antibody alone administration group, whereas in the NYZ-1010 and anti-mouse TIGIT antibody combination group, 78.3%, indicating that the antitumor effect was enhanced by the combination.
　図７７（２）。各単毒投与群では腫瘍が残存し、薬効が限定的である（それぞれ灰色の実線と細かい破線）のに対し、併用群１０例中２例の推定腫瘍体積は０ｍｍ３を示し、その後も完全退縮を維持した（黒丸、実線）。ＣＴ２６　ＮＹ－ＳＣＴを完全退縮マウスに再移植、及びＮａiｖｅマウス（雌性、１５～１７週齢）に移植した。ＣＴ２６　ＮＹ－ＳＣＴはＰＢＳ（Ｗａｋｏ社）で１×１０７細胞／ｍＬになるよう調製し、皮下に０．１ｍＬ移植した（Ｄａｙ５８）。Ｎａiｖｅマウスでは腫瘍が生着して増殖したが（粗い破線）、完全退縮マウスでは腫瘍が生着せず、ＮＹＺ－１０１０と抗マウスＴＩＧＩＴ抗体による完全退縮が維持され（黒丸、実線）、腫瘍細胞に対する免疫記憶が成立したことが示された。 Figure 77(2). In each monotoxin administration group, tumors remained and the drug efficacy was limited (gray solid line and thin broken line, respectively), whereas the estimated tumor volume in 2 out of 10 cases in the combination group was 0 mm3 , and even after Regression was maintained (black circles, solid lines). CT26 NY-SCT was reimplanted into completely regressed mice and into naive mice (female, 15-17 weeks old). CT26 NY-SCT was prepared at 1×10 7 cells/mL with PBS (Wako), and 0.1 mL was implanted subcutaneously (Day 58). Tumors engrafted and grew in naive mice (rough dashed line), but tumors did not engraft in mice with complete regression, and complete regression was maintained by NYZ-1010 and anti-mouse TIGIT antibody (black circles, solid line), and tumor cells It was shown that immunological memory was established.
実施例１３．　ヒト扁平上皮肺がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）とＣａｒｂｏｐｌａｔｉｎとのｉｎ　ｖｉｖｏ併用効果
　ヒト扁平上皮肺がん細胞株ＮＣＩ－Ｈ１７０３（ＡＴＣＣ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで６×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約１週間後（Ｄａｙ６～７）より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 13. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Carboplatin on human squamous cell lung cancer cell line NCI-H1703 (ATCC) ) was prepared at 6×10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm3) = Average value of estimated tumor volume for each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ１４に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、ヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５ｘ１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０２ ｍｇ／ｋｇ）を尾静脈内投与した。またＣａｒｂｏｐｌａｔｉｎ（ＴＣＩ社、５０ ｍｇ／ｋｇ）は腹腔内投与した。投与はＤａｙ１４、Ｄａｙ２１に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）、及びＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）は以下に示す計算式により算出した。 On Day 14, grouping was performed based on tumor volume so that n=5 in each group, and human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells to a concentration of 5x10 7 cells/mL in PBS. It was prepared and 0.2 mL was implanted into the tail vein. After 3-4 hours, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg/kg) was administered into the tail vein. Furthermore, Carboplatin (TCI, 50 mg/kg) was administered intraperitoneally. Administration was carried out on Day 14 and Day 21. Tumor Growth Inhibition (%) and Change from baseline (%) were calculated using the formulas shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、Ｃａｒｂｏｐｌａｔｉｎ単独投与、及びＮＹＺ－１０１０とＣａｒｂｏｐｌａｔｉｎ併用投与の結果を図７８に示した。移植３４日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で４７．２％、Ｃａｒｂｏｐｌａｔｉｎ単独投与群で１５.０％であったのに対し、ＮＹＺ－１０１０とＣａｒｂｏｐｌａｔｉｎ併用群では６１．６％であり、併用により抗腫瘍効果が増強されることが示された。 The results of NYZ-1010 administration alone, Carboplatin administration alone, and NYZ-1010 and Carboplatin combination administration are shown in FIG. 78. TGI 34 days after transplantation was 47.2% in the NYZ-1010 monotherapy group and 15.0% in the Carboplatin monotherapy group, whereas it was 61.6% in the NYZ-1010 and Carboplatin combination group. It was shown that the antitumor effect was enhanced by combined use.
実施例１４．　ヒト食道がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）とｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉyとのｉｎ　ｖｉｖｏ併用効果
　ヒト食道がん細胞株ＯＥ－１９（ＤＳＭＺ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで５×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約１週間後（Ｄａｙ６～７）より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 14. Effect of in vivo combined use of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and sacituzumab govitecan-hziy on human esophageal cancer cell line Human esophageal cancer cell line OE- 19 (DSMZ) was prepared in PBS containing 50% Matrigel (CORNING) to 5 x 10 7 cells/mL, and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). ). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm3) = Average value of estimated tumor volume for each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ６に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、ｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉy（２５ ｍｇ／ｋｇ）を尾静脈内投与した。Ｄａｙ７にヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５ｘ１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０１ ｍｇ／ｋｇ）を尾静脈内投与した。投与はＤａｙ７（ＮＹＺ－１０１０）、もしくはＤａｙ６、Ｄａｙ１３（ｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉy）に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）、及びＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）は以下に示す計算式により算出した。 On Day 6, the mice were divided into groups based on tumor volume so that n=5 in each group, and sacituzumab govitecan-hziy (25 mg/kg) was administered intravenously to the tail vein. On Day 7, human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells, which were prepared in PBS to a concentration of 5 x 10 7 cells/mL and 0.2 mL transplanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg/kg) was administered into the tail vein. Administration was performed on Day 7 (NYZ-1010), Day 6, or Day 13 (sacituzumab govitecan-hziy). Tumor Growth Inhibition (%) and Change from baseline (%) were calculated using the formulas shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００） TUMOR GROWTH INHIBITION ( %) = 100-
　ＮＹＺ－１０１０単独投与、ｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉy単独投与、及びＮＹＺ－１０１０とｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉy併用投与の結果を図７９（１）に示した。移植２１日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で７９．９％、ｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉy単独投与群で３５．４％であったのに対し、ＮＹＺ－１０１０とｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉy併用群では９６．７％であり、併用により抗腫瘍効果が増強されることが示された。またＤａｙ６の腫瘍体積をベースラインとした各個体のＤａｙ２１の腫瘍体積の変化率Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）を以下に示す計算式により算出し、図７９（２）に示した。また各群のＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）平均値を表５に記載した。ＮＹＺ－１０１０とｓａｃｉｔｕｚｕｍａｂ　ｇｏｖｉｔｅｃａｎ－ｈｚｉyの併用による腫瘍退縮効果が示された。 The results of the single administration of NYZ-1010, the single administration of sacituzumab govitecan-hziy, and the combined administration of NYZ-1010 and sacituzumab govitecan-hziy are shown in FIG. 79 (1). TGI 21 days after transplantation was 79.9% in the NYZ-1010 monotherapy group and 35.4% in the sacituzumab govitecan-hziy monotherapy group, whereas in the NYZ-1010 and sacituzumab govitecan-hziy combination therapy group 96 in group .7%, indicating that the antitumor effect was enhanced by combined use. Furthermore, the rate of change from baseline (%) in the tumor volume on Day 21 of each individual with the tumor volume on Day 6 as the baseline was calculated using the formula shown below, and is shown in FIG. 79 (2). Furthermore, the average value of Change from baseline (%) for each group is listed in Table 5. The tumor regression effect of the combination of NYZ-1010 and sacituzumab govitecan-hziy was demonstrated.
　Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）＝（Ｄａｙ２１におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ－Ｄａｙ６におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ）／Ｄａｙ６におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００
  Change from baseline (%) = (Estimated Tumor Volume on Day 21 - Estimated Tumor Volume on Day 6) / Estimated Tumor Volume on Day 6 x 100
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
実施例１５．　ヒト肺腺がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）とＤｏｘｏｒｕｂｉｃｉｎとのｉｎ　ｖｉｖｏ併用効果
　ヒト滑膜肉腫細胞株ＳＷ９８２（ＡＴＣＣ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで５×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約１週間後（Ｄａｙ６～７）より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 15. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Doxorubicin on human lung adenocarcinoma cell line Human synovial sarcoma cell line SW982 (ATCC) was prepared in PBS containing 50% Matrigel (CORNING) to a concentration of 5×10 7 cells/mL, and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］ Estimated Tumor Volume (mm3) = Average value of estimated tumor volume for each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ４０に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、Ｄｏｘｏｒｕｂｉｃｉｎ（サンド社、２．５ ｍｇ／ｋｇ）を尾静脈内投与した。Ｄａｙ４１にヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５ｘ１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０１ ｍｇ／ｋｇ）を尾静脈内投与した。投与はＤａｙ４１（ＮＹＺ－１０１０）、もしくはＤａｙ４０、Ｄａｙ４７（Ｄｏｘｏｒｕｂｉｃｉｎ）に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）、及びＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）は以下に示す計算式により算出した。 On Day 40, the mice were divided into groups based on tumor volume so that n=5 in each group, and Doxorubicin (Sandoz, 2.5 mg/kg) was administered into the tail vein. On Day 41, human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) and proliferated T cells were prepared in PBS to 5x10 7 cells/mL and 0.2 mL was transplanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg/kg) was administered into the tail vein. Administration was performed on Day 41 (NYZ-1010), Day 40, and Day 47 (Doxorubicin). Tumor Growth Inhibition (%) and Change from baseline (%) were calculated using the formulas shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００）
 
　ＮＹＺ－１０１０単独投与、Ｄｏｘｏｒｕｂｉｃｉｎ単独投与、及びＮＹＺ－１０１０とＤｏｘｏｒｕｂｉｃｉｎ併用投与の結果を図８０（１）に示した。移植５５日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で７１．８％、Ｄｏｘｏｒｕｂｉｃｉｎ単独投与群で２５．６％であったのに対し、ＮＹＺ－１０１０とＤｏｘｏｒｕｂｉｃｉｎ併用群では９０．２％であり、併用により抗腫瘍効果が増強されることが示された。またＤａｙ４０の腫瘍体積をベースラインとした各個体のＤａｙ５５の腫瘍体積の変化率Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）を以下に示す計算式により算出し、図８０（２）に示した。また各群のＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）平均値を表６に記載した。ＮＹＺ－１０１０とＤｏｘｏｒｕｂｉｃｉｎの併用による腫瘍退縮効果が示された。 Tumor Growth Inhibition (%) = 100 - (Estimated Tumor Volume of each group/Estimated Tumor Volume of Vehicle Control x 100)

The results of NYZ-1010 administration alone, Doxorubicin administration alone, and NYZ-1010 and Doxorubicin combination administration are shown in FIG. 80(1). TGI 55 days after transplantation was 71.8% in the NYZ-1010 alone administration group and 25.6% in the Doxorubicin alone administration group, whereas it was 90.2% in the NYZ-1010 and Doxorubicin combination group. It was shown that the antitumor effect was enhanced by combined use. Furthermore, the rate of change from baseline (%) in the tumor volume on Day 55 of each individual with the tumor volume on Day 40 as the baseline was calculated using the formula shown below, and is shown in FIG. 80 (2). Furthermore, the average value of Change from baseline (%) for each group is listed in Table 6. A tumor regression effect was demonstrated by the combination of NYZ-1010 and Doxorubicin.
　Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）＝（Ｄａｙ５５におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ－Ｄａｙ４０におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ）／Ｄａｙ４０におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００ Change from baseline (%) = (Estimated Tumor Volume at Day 55 - Estimated Tumor Volume at Day 40) / Estimated Tumor Volume at Day 40 r　Volume×100
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
実施例１６．　ヒト扁平上皮肺がん細胞株に対するＦｃ付加型抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０）とＤｅｃｉｔａｂｉｎｅとのｉｎ　ｖｉｖｏ併用効果
　ヒト扁平上皮肺がん細胞株ＮＣＩ－Ｈ１７０３（ＡＴＣＣ）を５０％Ｍａｔｒｉｇｅｌ（ＣＯＲＮＩＮＧ社）含有ＰＢＳで６×１０７細胞／ｍＬになるよう調製し、ＮＳＧマウス（雌性、４～６週齢）の皮下に０．１ｍＬ移植した（Ｄａｙ ０）。約１週間後（Ｄａｙ６～７）より経時的に腫瘍の長径（ｍｍ）及び短径（ｍｍ）を電子デジタルノギスで計測し、以下に示す計算式により推定腫瘍体積を算出した。 Example 16. In vivo combined effect of Fc-added anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Decitabine on human squamous cell lung cancer cell line Human squamous cell lung cancer cell line NCI-H1703 (ATCC ) was prepared at 6×10 7 cells/mL in PBS containing 50% Matrigel (CORNING), and 0.1 mL was subcutaneously transplanted into NSG mice (female, 4 to 6 weeks old) (Day 0). Approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below.
　　Ｅｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ（ｍｍ３）＝各個体の推定腫瘍体積の平均値：
　　各個体の推定腫瘍体積（ｍｍ３）＝１／２×［腫瘍長径］×［腫瘍短径］×［腫瘍短径］
  Estimated Tumor Volume (mm3) = Average value of estimated tumor volume for each individual:
Estimated tumor volume of each individual (mm 3 ) = 1/2 × [Tumor major axis] × [Tumor minor axis] × [Tumor minor axis]
　Ｄａｙ７に各群ｎ＝５になるよう腫瘍体積による群分けを実施し、Ｄｅｃｉｔａｂｉｎｅ（東京化成工業株式会社、３ ｍｇ／ｋｇ）を尾静脈内投与した。Ｄａｙ１４にヒトＰＢＭＣをＣＤ３／ＣＤ２８ Ｄｙｎａｂｅａｄｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）により刺激して増殖させたＴ細胞をＰＢＳで５ｘ１０７細胞／ｍＬになるよう調製し、０．２ｍＬ尾静脈内移植した。３－４時間後に抗ＨＬＡ－Ａ２／ＮＹ－ＥＳＯ－抗ＣＤ３二重特異性分子（ＮＹＺ－１０１０、０．０２ ｍｇ／ｋｇ）を尾静脈内投与した。投与はＤａｙ１４、Ｄａｙ２１（ＮＹＺ－１０１０）、もしくはＤａｙ７、Ｄａｙ９、Ｄａｙ１１（Ｄｅｃｉｔａｂｉｎｅ）に実施した。Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）、及びＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）は以下に示す計算式により算出した。 On Day 7, the mice were divided into groups based on tumor volume so that n=5 in each group, and Decitabine (Tokyo Kasei Kogyo Co., Ltd., 3 mg/kg) was administered into the tail vein. On Day 14, human PBMC were stimulated with CD3/CD28 Dynabeads (ThermoFisher) to proliferate T cells, which were prepared in PBS to a concentration of 5 x 10 7 cells/mL, and 0.2 mL was transplanted into the tail vein. 3-4 hours later, anti-HLA-A2/NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg/kg) was administered into the tail vein. Administration was performed on Day 14, Day 21 (NYZ-1010), or Day 7, Day 9, Day 11 (Decitabine). Tumor Growth Inhibition (%) and Change from baseline (%) were calculated using the formulas shown below.
　　Ｔｕｍｏｒ　Ｇｒｏｗｔｈ　Ｉｎｈｉｂｉｔｉｏｎ（％）＝１００－（各群のＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ／Ｖｅｈｉｃｌｅ　ＣｏｎｔｒｏｌのＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００）
 
　Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）＝（Ｄａｙ３２におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ－Ｄａｙ７におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ）／Ｄａｙ７におけるＥｓｔｉｍａｔｅｄ　Ｔｕｍｏｒ　Ｖｏｌｕｍｅ×１００
  Tumor Growth Inhibition (%) = 100 - (Estimated Tumor Volume of each group/Estimated Tumor Volume of Vehicle Control x 100)

Change from baseline (%) = (Estimated Tumor Volume on Day 32 - Estimated Tumor Volume on Day 7) / Estimated Tumor Volume on Day 7 x 100
　ＮＹＺ－１０１０単独投与、Ｄｅｃｉｔａｂｉｎｅ単独投与、及びＮＹＺ－１０１０とＤｅｃｉｔａｂｉｎｅ併用投与の結果を図８１（１）に示した。移植３２日後におけるＴＧＩは、ＮＹＺ－１０１０単独投与群で６８．０％、Ｄｅｃｉｔａｂｉｎｅ単独投与群で４６．７％であったのに対し、ＮＹＺ－１０１０とＤｅｃｉｔａｂｉｎｅ併用群では９３．０％であり、併用により抗腫瘍効果が増強されることが示された。
またＤａｙ７の腫瘍体積をベースラインとした各個体のＤａｙ３２の腫瘍体積の変化率Ｃｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）を以下に示す計算式により算出し、図８１（２）に示した。また各群のＣｈａｎｇｅ　ｆｒｏｍ　ｂａｓｅｌｉｎｅ（％）平均値を表７に記載した。 The results of NYZ-1010 administration alone, Decitabine administration alone, and NYZ-1010 and Decitabine combination administration are shown in FIG. 81 (1). TGI 32 days after transplantation was 68.0% in the NYZ-1010 alone administration group and 46.7% in the Decitabine alone administration group, whereas it was 93.0% in the NYZ-1010 and Decitabine combination group, It was shown that the antitumor effect was enhanced by combined use.
Furthermore, the rate of change from baseline (%) in the tumor volume on Day 32 for each individual with the tumor volume on Day 7 as the baseline was calculated using the formula shown below, and is shown in FIG. 81 (2). In addition, the average value of Change from baseline (%) for each group is listed in Table 7.
Figure JPOXMLDOC01-appb-T000007
  
Figure JPOXMLDOC01-appb-T000007
  
　本発明の提供する抗体と他剤の組合せにより各種癌の治療または予防が可能となる。 The combination of the antibody provided by the present invention and other drugs enables the treatment or prevention of various cancers.
配列番号１：ＮＹＡ－０００１に含まれるＣＤＲＨ１のアミノ酸配列
配列番号２：ＮＹＡ－０００１に含まれるＣＤＲＨ２のアミノ酸配列
配列番号３：欠番（２０２２年３月１６日出願した特願２０２２－０４１２５３の配列番号３はＮＹＡ－０００１に含まれるＣＤＲＨ３のアミノ酸配列であった）
配列番号４：ＮＹＡ－０００１に含まれるＣＤＲＬ１のアミノ酸配列
配列番号５：ＮＹＡ－０００１に含まれるＣＤＲＬ２のアミノ酸配列
配列番号６：ＮＹＡ－０００１に含まれるＣＤＲＬ３のアミノ酸配列
配列番号７：Ｃ３Ｅ－７０８５の重鎖ＣＤＲ１のアミノ酸配列
配列番号８：Ｃ３Ｅ－７０８５の重鎖ＣＤＲ３のアミノ酸配列
配列番号９：Ｃ３Ｅ－７０８５の重鎖ＣＤＲ４のアミノ酸配列
配列番号１０：Ｃ３Ｅ－７０８５の軽鎖ＣＤＲ１のアミノ酸配列
配列番号１１：欠番（２０２２年３月１６日出願の優先権の配列番号１１はＣ３Ｅ－７０８５の軽鎖ＣＤＲ２のアミノ酸配列であった）
配列番号１２：Ｃ３Ｅ－７０８５の軽鎖ＣＤＲ３のアミノ酸配列
配列番号１３：ＮＹＡ－０００１の重鎖可変領域のアミノ酸配列
配列番号１４：ＮＹＡ－０００１の軽鎖可変領域のアミノ酸配列
配列番号１５：ＮＹＡ－００８２の重鎖可変領域のアミノ酸配列
配列番号１６：ＮＹＡ－００８２の軽鎖可変領域のアミノ酸配列
配列番号１７：ＮＹＡ－１１６３全長のアミノ酸配列
配列番号１８：ＮＹＡ－２０２３全長のアミノ酸配列
配列番号１９：ＮＹＡ－２０２７全長のアミノ酸配列
配列番号２０：ＮＹＡ－１１４３全長のアミノ酸配列
配列番号２１：ＮＹＡ－１１４３全長のアミノ酸配列
配列番号２２：ＮＹＡ－２１４３全長のアミノ酸配列
配列番号２３：ＮＹＡ－１１４３－ＶＬ０１のアミノ酸配列
配列番号２４：ＮＹＡ－２０４４全長のアミノ酸配列
配列番号２５： ＮＹＡ－２０４５全長のアミノ酸配列
配列番号２６：ＮＹＡ－２０４７全長のアミノ酸配列
配列番号２７：ＮＹＡ－２０４８全長のアミノ酸配列
配列番号２８：ＮＹＡ－２０６０全長のアミノ酸配列
配列番号２９：ＮＹＡ－２０６１全長のアミノ酸配列
配列番号３０：ＮＹＡ－０００１全長のアミノ酸配列
配列番号３１：ＨＣ１全長のアミノ酸配列
配列番号３２：ＮＹＦ－００１６－ＨＣ２全長のアミノ酸配列
配列番号３３：ＮＹＦ－００１９－ＨＣ２全長のアミノ酸配列
配列番号３４：ＮＹＦ－００２２－ＨＣ２全長のアミノ酸配列
配列番号３５：ＮＹＦ－００２３－ＨＣ２全長のアミノ酸配列
配列番号３６：ＮＹＦ－００２７－ＨＣ２全長のアミノ酸配列
配列番号３７：ＮＹＦ－００３５－ＨＣ２全長のアミノ酸配列
配列番号３８：ＮＹＦ－００４４－ＨＣ２全長のアミノ酸配列
配列番号３９：ＮＹＦ－００４５－ＨＣ２全長のアミノ酸配列
配列番号４０：ＮＹＦ－００４７－ＨＣ２全長のアミノ酸配列
配列番号４１：ＮＹＦ－００４８－ＨＣ２全長のアミノ酸配列
配列番号４２：ＮＹＦ－００６０－ＨＣ２全長のアミノ酸配列
配列番号４３：ＮＹＦ－００６１－ＨＣ２全長のアミノ酸配列
配列番号４４：ＮＹＡ－０００１－Ｆａｂ－ＨＣ１－κ　ｄｅｌｅｔｅ全長のアミノ酸配列
配列番号４５：ＮＹＡ－０００１－ＬＣ全長のアミノ酸配列
配列番号４６：Ｃ３Ｅ－７０３４のアミノ酸配列
配列番号４７：Ｃ３Ｅ－７０３６のアミノ酸配列
配列番号４８：Ｃ３Ｅ－７０８５のアミノ酸配列
配列番号４９：Ｃ３Ｅ－７０８８のアミノ酸配列
配列番号５０：Ｃ３Ｅ－７０９３のアミノ酸配列
配列番号５１：Ｃ３Ｅ－７０７８のアミノ酸配列
配列番号５２：ＮＹＦ－００１４－ＨＣ２全長のアミノ酸配列
配列番号５３：ＮＹＦ－００８２－ＨＣ２全長のアミノ酸配列
配列番号５４：ＮＹＦ－００８３－ＨＣ２全長のアミノ酸配列
配列番号５５：ＮＹＺ－００８２－ＨＣ２全長のアミノ酸配列
配列番号５６：ＮＹＺ－００８３－ＨＣ２全長のアミノ酸配列
配列番号５７：ＮＹＺ－１０１０－ＨＣ２全長のアミノ酸配列
配列番号５８：Ｃ３Ｅ－７０８５－ＬＣ全長のアミノ酸配列
配列番号５９：Ｃ３Ｅ－７０８５　ｓｃＦｖ全長アミノ酸配列
配列番号６０：ＮＹＺ－１００７－ＨＣ２全長のアミノ酸配列
配列番号６１：ＮＹＺ－１０１７－ＨＣ２全長のアミノ酸配列
配列番号６２：Ｃ３Ｅ－７０９６全長アミノ酸配列
配列番号６３：Ｃ３Ｅ－７０９７全長アミノ酸配列
配列番号６４：Ｃ３Ｅ－７０９８全長アミノ酸配列
配列番号６５：Ｃ３Ｅ－７０９９全長アミノ酸配列
配列番号６６：ＮＹ－ＥＳＯのアミノ酸配列
配列番号６７：ＨＬＡ－Ａ＊０２０１（ＧｅｎＢａｎｋ：ＡＳＡ４７５３４．１）のトランケート体のアミノ酸配列
配列番号６８：β２－マイクログロブリンのアミノ酸配列
配列番号６９：ＮＹＡ－１１４３－ＶＨ０２のアミノ酸配列
配列番号７０：ＮＹＡ－１１４３－ＶＨ０３のアミノ酸配列
配列番号７１：ＮＹＡ－１１５４全長のアミノ酸配列 SEQ ID NO: 1: Amino acid sequence of CDRH1 contained in NYA-0001 SEQ ID NO: 2: Amino acid sequence of CDRH2 contained in NYA-0001 SEQ ID NO: 3: Missing number (SEQ ID NO: of patent application 2022-041253 filed on March 16, 2022) 3 was the amino acid sequence of CDRH3 contained in NYA-0001)
SEQ ID NO: 4: Amino acid sequence of CDRL1 contained in NYA-0001 SEQ ID NO: 5: Amino acid sequence of CDRL2 contained in NYA-0001 SEQ ID NO: 6: Amino acid sequence of CDRL3 contained in NYA-0001 SEQ ID NO: 7: of C3E-7085 Amino acid sequence of heavy chain CDR1 of C3E-7085 SEQ ID NO: 8: Amino acid sequence of heavy chain CDR3 of C3E-7085 SEQ ID NO: 9: Amino acid sequence of heavy chain CDR4 of C3E-7085 SEQ ID NO: 10: Amino acid sequence of light chain CDR1 of C3E-7085 SEQ ID NO: 11: Missing number (SEQ ID NO: 11 of the priority application filed on March 16, 2022 was the amino acid sequence of C3E-7085 light chain CDR2)
SEQ ID NO: 12: Amino acid sequence of light chain CDR3 of C3E-7085 SEQ ID NO: 13: Amino acid sequence of heavy chain variable region of NYA-0001 SEQ ID NO: 14: Amino acid sequence of light chain variable region of NYA-0001 SEQ ID NO: 15: NYA- Amino acid sequence of heavy chain variable region of 0082 SEQ ID NO: 16: Amino acid sequence of light chain variable region of NYA-0082 SEQ ID NO: 17: NYA-1163 full-length amino acid sequence SEQ ID NO: 18: NYA-2023 full-length amino acid sequence SEQ ID NO: 19: NYA-2027 full-length amino acid sequence SEQ ID NO: 20: NYA-1143 full-length amino acid sequence SEQ ID NO: 21: NYA-1143 full-length amino acid sequence SEQ ID NO: 22: NYA-2143 full-length amino acid sequence SEQ ID NO: 23: NYA-1143-VL01 Amino acid sequence SEQ ID NO: 24: NYA-2044 full-length amino acid sequence SEQ ID NO: 25: NYA-2045 full-length amino acid sequence SEQ ID NO: 26: NYA-2047 full-length amino acid sequence SEQ ID NO: 27: NYA-2048 full-length amino acid sequence SEQ ID NO: 28: NYA-2060 full-length amino acid sequence SEQ ID NO: 29: NYA-2061 full-length amino acid sequence SEQ ID NO: 30: NYA-0001 full-length amino acid sequence SEQ ID NO: 31: HC1 full-length amino acid sequence SEQ ID NO: 32: NYF-0016-HC2 full-length amino acid sequence SEQ ID NO: 33: NYF-0019-HC2 full-length amino acid sequence SEQ ID NO: 34: NYF-0022-HC2 full-length amino acid sequence SEQ ID NO: 35: NYF-0023-HC2 full-length amino acid sequence SEQ ID NO: 36: NYF-0027-HC2 full-length Amino acid sequence SEQ ID NO: 37: NYF-0035-HC2 full-length amino acid sequence SEQ ID NO: 38: NYF-0044-HC2 full-length amino acid sequence SEQ ID NO: 39: NYF-0045-HC2 full-length amino acid sequence SEQ ID NO: 40: NYF-0047- HC2 full-length amino acid sequence SEQ ID NO: 41: NYF-0048-HC2 full-length amino acid sequence SEQ ID NO: 42: NYF-0060-HC2 full-length amino acid sequence SEQ ID NO: 43: NYF-0061-HC2 full-length amino acid sequence SEQ ID NO: 44: NYA- 0001-Fab-HC1-κ delete full-length amino acid sequence SEQ ID NO: 45: NYA-0001-LC full-length amino acid sequence SEQ ID NO: 46: C3E-7034 amino acid sequence SEQ ID NO: 47: C3E-7036 amino acid sequence SEQ ID NO: 48: C3E -7085 amino acid sequence SEQ ID NO: 49: C3E-7088 amino acid sequence SEQ ID NO: 50: C3E-7093 amino acid sequence SEQ ID NO: 51: C3E-7078 amino acid sequence SEQ ID NO: 52: NYF-0014-HC2 full length amino acid sequence SEQ ID NO: 53: NYF-0082-HC2 full-length amino acid sequence SEQ ID NO: 54: NYF-0083-HC2 full-length amino acid sequence SEQ ID NO: 55: NYZ-0082-HC2 full-length amino acid sequence SEQ ID NO: 56: NYZ-0083-HC2 full-length amino acid sequence SEQ ID NO: 57: NYZ-1010-HC2 full-length amino acid sequence SEQ ID NO: 58: C3E-7085-LC full-length amino acid sequence SEQ ID NO: 59: C3E-7085 scFv full-length amino acid sequence SEQ ID NO: 60: NYZ-1007-HC2 full-length amino acid sequence SEQ ID NO: 61: NYZ-1017-HC2 full-length amino acid sequence SEQ ID NO: 62: C3E-7096 full-length amino acid sequence SEQ ID NO: 63: C3E-7097 full-length amino acid sequence SEQ ID NO: 64: C3E-7098 full-length amino acid sequence SEQ ID NO: 65: C3E-7099 Full-length amino acid sequence SEQ ID NO: 66: NY-ESO amino acid sequence SEQ ID NO: 67: HLA-A*0201 (GenBank: ASA47534.1) truncated amino acid sequence SEQ ID NO: 68: β2-microglobulin amino acid sequence SEQ ID NO: 69: Amino acid sequence of NYA-1143-VH02 SEQ ID NO: 70: Amino acid sequence of NYA-1143-VH03 SEQ ID NO: 71: NYA-1154 full-length amino acid sequence

Claims (64)

  1. 　下記［ｉｉ］記載の化合物を組み合わせて使用される、下記［ｉ］記載の多重特異性抗体を含むがんの治療及び／又は予防のための医薬組成物：
    ［ｉ］
    　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、若しくは配列番号３で表されるアミノ酸配列において、６番目のアミノ酸がＮであるアミノ酸配列からなる軽鎖ＣＤＲＨ３、
    配列番号４で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、若しくは配列番号４で表されるアミノ酸配列において、７番目のアミノ酸がＷ及び／若しくは８番目のアミノ酸がＫであるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、並びに
    配列番号６で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３、若しくは配列番号６で表されるアミノ酸配列において、２番目のアミノ酸がＡ若しくはＳであるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    を含む、ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片、
    並びに、
    　配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２若しくは配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＮ又はＳであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２若しくはＡｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＧ、Ｑ、Ｎ、Ｓ又はＡであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、並びに
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    を含む、ＣＤ３に結合する抗体又はその抗原結合断片、
    を含む、多重特異性抗体；
    ［ｉｉ］
    　免疫チェックポイント分子を阻害する化合物、又は、化学療法剤。     A pharmaceutical composition for the treatment and/or prevention of cancer, comprising the multispecific antibody described in [i] below, which is used in combination with the compound described in [ii] below:
    [i]
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
    A heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, or a light chain CDRH3 consisting of an amino acid sequence in which the 6th amino acid is N in the amino acid sequence represented by SEQ ID NO: 3,
    A light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, or a light chain consisting of an amino acid sequence in which the 7th amino acid is W and/or the 8th amino acid is K in the amino acid sequence represented by SEQ ID NO: 4. CDRL1,
    Light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of Figure 11: SEQ ID NO: 5 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, or A light chain CDRL3 consisting of an amino acid sequence represented by SEQ ID NO: 6 in which the second amino acid is A or S.
    an antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO,
    and,
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8 or heavy chain CDRH2 consisting of the amino acid sequence in which the third amino acid is N or S in the amino acid sequence represented by SEQ ID NO: 8,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top in Figure 12: SEQ ID NO: 11 in Figure 82) or Arg Asp Asp (fifth sequence from the top in Figure 12: SEQ ID NO: 11 in Figure 82) A light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 11) in which the second amino acid is G, Q, N, S or A, and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 12. CDRL3
    an antibody that binds to CD3 or an antigen-binding fragment thereof,
    multispecific antibodies, including;
    [ii]
    Compounds that inhibit immune checkpoint molecules or chemotherapeutic agents.
  2. 　多重特異性抗体が二重特異性抗体である、請求項１記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the multispecific antibody is a bispecific antibody.
  3. 　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片が、下記（ｉ）～（ｖ）のセットからなる群より選択される１つ又は２つ以上のセットのＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含む、請求項１又は２記載の医薬組成物：
    （ｉ）
    　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号４で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号６で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    （ｉｉ）
    　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号４で表されるアミノ酸配列において、７番目のアミノ酸がＷであるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号６で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３

    （ｉｉｉ）
    　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号３で表されるアミノ酸配列において、６番目のアミノ酸がＮであるアミノ酸配からなる重鎖ＣＤＲＨ３、
    配列番号４で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号６で表されるアミノ酸配列において、２番目のアミノ酸がＡであるアミノ酸配列からなる軽鎖ＣＤＲＬ３、
    （ｉｖ）
    　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号４で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号６で表されるアミノ酸配列において、２番目のアミノ酸がＳであるアミノ酸配列からなる軽鎖ＣＤＲＬ３、並びに
    　（ｖ）
    　配列番号１で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号２で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号３で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号４で表されるアミノ酸配列において、７番目のアミノ酸がＷであり、８番目のアミノ酸がＫであるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｓｐ　Ａｓｎ　Ａｓｎ（図１１の上から５番目の配列：図８２の配列番号５）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号６で表されるアミノ酸配列において、２番目のアミノ酸がＡであるアミノ酸配列からなる軽鎖ＣＤＲＬ３。     The antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO binds one or more sets of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following sets (i) to (v). The pharmaceutical composition according to claim 1 or 2, comprising:
    (i)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4,
    A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6
    (ii)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, where the seventh amino acid is W;
    A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6

    (iii)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
    Heavy chain CDRH3 consisting of an amino acid sequence in which the 6th amino acid is N in the amino acid sequence represented by SEQ ID NO: 3;
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4,
    A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, where the second amino acid is A;
    (iv)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4,
    A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
    A light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, in which the second amino acid is S, and (v)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3,
    Light chain CDRL1 consisting of an amino acid sequence in which the seventh amino acid is W and the eighth amino acid is K in the amino acid sequence represented by SEQ ID NO: 4,
    A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (fifth sequence from the top of FIG. 11: SEQ ID NO: 5 in FIG. 82), and
    A light chain CDRL3 consisting of an amino acid sequence represented by SEQ ID NO: 6, in which the second amino acid is A.
  4. 　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片が、配列番号１８で表されるアミノ酸配列のアミノ酸番号２１～１４０のアミノ酸配列、又は配列番号６９若しくは配列番号７０で表されるアミノ酸配列と９５％以上の配列同一性を有するアミノ酸配列からなる重鎖可変領域、及び、配列番号１８若しくは配列番号２８で表されるアミノ酸配列のアミノ酸番号１５６～２６６のアミノ酸配列、又は配列番号２３で表されるアミノ酸配列と９５％以上の配列同一性を有するアミノ酸配列からなる軽鎖可変領域を含む、請求項１～３のいずれか一つに記載の医薬組成物。 The antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO has the amino acid sequence of amino acids 21 to 140 of the amino acid sequence represented by SEQ ID NO: 18, or the amino acid sequence represented by SEQ ID NO: 69 or SEQ ID NO: 70. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity, and an amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 18 or SEQ ID NO: 28, or the amino acid sequence represented by SEQ ID NO: 23. The pharmaceutical composition according to any one of claims 1 to 3, comprising a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence.
  5. 　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片が、
    　配列番号１３で表されるアミノ酸配列からなる重鎖可変領域
    配列番号１５で表されるアミノ酸配列からなる重鎖可変領域、
    配列番号２０で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号１７で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号１８で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号１９で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号２２で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号２４で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号２５で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号２６で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号２７で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号２８で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号２９で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号２１で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、
    配列番号５５で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、若しくは、
    　配列番号７１で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域、並びに
    配列番号１４で表されるアミノ酸配列からなる軽鎖可変領域、
    配列番号１６で表されるアミノ酸配列からなる軽鎖可変領域、
    配列番号２０で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号１７で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号１８で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号１９で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２２で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２４で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２５で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２６で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２７で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２８で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２９で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２１で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５５で表されるアミノ酸配列の１６１番目～２７１番目のアミノ酸配列からなる軽鎖可変領域、若しくは、
    　配列番号７１で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域を含む、請求項１乃至４のいずれか一つに記載の医薬組成物。     An antibody that binds to HLA/NY-ESO or an antigen-binding fragment thereof,
    A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13, a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 14,
    A light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29,
    A light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21,
    A light chain variable region consisting of the 161st to 271st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or
    The pharmaceutical composition according to any one of claims 1 to 4, comprising a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71.
  6. 　配列番号１３で表されるアミノ酸配列からなる重鎖可変領域及び配列番号１４で表されるアミノ酸配列からなる軽鎖可変領域、
    配列番号１５で表されるアミノ酸配列からなる重鎖可変領域及び配列番号１６で表されるアミノ酸配列からなる軽鎖可変領域、
    配列番号２０で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２０で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号１７で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号１７で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号１８で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号１８で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号１９で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号１９で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２２で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２２で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２４で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２４で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２５で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２５で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２６で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２６で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２７で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２７で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２８で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２８で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２９で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２９で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号２１で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号２１で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域、配列番号５５で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号５５で表されるアミノ酸配列の１６１番目～２７１番目のアミノ酸配列からなる軽鎖可変領域、又は、
    配列番号７１で表されるアミノ酸配列の２１番目～１４０番目のアミノ酸配列からなる重鎖可変領域及び配列番号７１で表されるアミノ酸配列の１５６番目～２６６番目のアミノ酸配列からなる軽鎖可変領域を含む、請求項１～５記載のいずれか一つに記載の医薬組成物。     A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 14,
    A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29,
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21, and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21, A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, and a light chain variable region consisting of the 161st to 271st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, Or
    A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71. The pharmaceutical composition according to any one of claims 1 to 5, comprising:
  7. 　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片がｓｃＦｖである、請求項１～６のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO is an scFv.
  8. 　ＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片が、
    　配列番号３０で表されるアミノ酸配列の２１～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号１５で表されるアミノ酸配列からなる重鎖可変領域及び配列番号１６で表されるアミノ酸配列からなる軽鎖可変領域を含むｓｃＦｖ、
    配列番号２０で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号１７で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号１８で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号１９で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号２２で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号２４で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号２５で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号２６で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号２７で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号２８で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号２９で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号２１で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖ、配列番号５５で表されるアミノ酸配列の２１番目～２７１番目のアミノ酸配列からなるｓｃＦｖ、又は、
    　配列番号７１で表されるアミノ酸配列の２１番目～２６６番目のアミノ酸配列からなるｓｃＦｖである、請求項１～７のいずれか一つに記載の医薬組成物。     An antibody that binds to HLA/NY-ESO or an antigen-binding fragment thereof,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 30,
    scFv comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 17,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 19,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 25,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 26,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 27,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 28,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 29,
    scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 21, scFv consisting of the 21st to 271st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or
    The pharmaceutical composition according to any one of claims 1 to 7, which is an scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 71.
  9. 　ＣＤ３に結合する抗体又はその抗原結合断片が、下記（ｉ）～（X）のセットからなる群より選択される１つ又は２つ以上のセットのＣＤＲＨ１～ＣＤＲＨ３及びＣＤＲＬ１～ＣＤＲＬ３を含む、請求項１～８のいずれか一つに記載の医薬組成物：
    （ｉ）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    （ｉｉ）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＮであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    （ｉｉｉ）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＳであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    （ｉｖ）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＧであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    （ｖ）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＱであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    （ｖｉ）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＮであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    （ｖｉｉ）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＳであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３、
    （ｖｉｉｉ）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＡであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３、
    （ｉX）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＳであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＮであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３
    並びに
    （X）
    配列番号７で表されるアミノ酸配列からなる重鎖ＣＤＲＨ１、
    配列番号８で表されるアミノ酸配列において、３番目のアミノ酸がＳであるアミノ酸配列からなる重鎖ＣＤＲＨ２、
    配列番号９で表されるアミノ酸配列からなる重鎖ＣＤＲＨ３、
    配列番号１０で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ１、
    Ａｒｇ　Ａｓｐ　Ａｓｐ（図１２の上から５番目の配列：図８２の配列番号１１）で表されるアミノ酸配列において、２番目のアミノ酸がＳであるアミノ酸配列からなる軽鎖ＣＤＲＬ２、及び、
    配列番号１２で表されるアミノ酸配列からなる軽鎖ＣＤＲＬ３。     A claim in which the antibody or antigen-binding fragment thereof that binds to CD3 comprises one or more sets of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following sets (i) to (X): Pharmaceutical composition according to any one of 1 to 8:
    (i)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
    (ii)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is N in the amino acid sequence represented by SEQ ID NO: 8;
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
    (iii)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8;
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    Light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), and light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
    (iv)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    In the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of Figure 12: SEQ ID NO: 11 in Figure 82), the light chain CDRL2 consists of an amino acid sequence in which the second amino acid is G, and SEQ ID NO: 12. Light chain CDRL3 consisting of the amino acid sequence shown
    (v)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    A light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), in which the second amino acid is Q, and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
    (vi)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    A light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), where the second amino acid is N, and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
    (vii)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    In the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 of FIG. 82), a light chain CDRL2 consisting of an amino acid sequence in which the second amino acid is S, and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12,
    (viii)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8,
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    A light chain CDRL2 consisting of an amino acid sequence in which the second amino acid is A in the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12,
    (iX)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8;
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    A light chain CDRL2 consisting of an amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 in FIG. 82), where the second amino acid is N, and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12
    and (X)
    Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7,
    Heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid is S in the amino acid sequence represented by SEQ ID NO: 8;
    Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9,
    Light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10,
    In the amino acid sequence represented by Arg Asp Asp (fifth sequence from the top of FIG. 12: SEQ ID NO: 11 of FIG. 82), a light chain CDRL2 consisting of an amino acid sequence in which the second amino acid is S, and
    Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.
  10. 　ＣＤ３に結合する抗体又はその抗原結合断片が、
    配列番号４６で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
    配列番号４７で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
    配列番号５１で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
    配列番号４８で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
    配列番号４９で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
    配列番号５０で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域、
    配列番号５４で表されるアミノ酸配列の２７２番目～３８９番目のアミノ酸配列からなる重鎖可変領域、
    配列番号５５で表されるアミノ酸配列の２７７番目～３９４番目のアミノ酸配列からなる重鎖可変領域、若しくは
    配列番号５６で表されるアミノ酸配列の２７７番目～３９４番目のアミノ酸配列からなる重鎖可変領域、並びに、
    配列番号４６で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号４７で表されるアミノ酸配列の１３５番目～２４１番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５１で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号４８で表されるアミノ酸配列の１３５番目～２４１番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号４９で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５０で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５４で表されるアミノ酸配列の４０５番目～５１１番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５５で表されるアミノ酸配列の４１０番目～５１６番目のアミノ酸配列からなる軽鎖可変領域、若しくは
    配列番号５６で表されるアミノ酸配列の４１０番目～５１６番目のアミノ酸配列からなる軽鎖可変領域、
    を含む、請求項９に記載の医薬組成物。     An antibody that binds to CD3 or an antigen-binding fragment thereof,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50,
    A heavy chain variable region consisting of the 272nd to 389th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54,
    A heavy chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or a heavy chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56. ,and,
    A light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46,
    A light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47,
    A light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51,
    A light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48,
    A light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49,
    A light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50,
    A light chain variable region consisting of the 405th to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54,
    A light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56. ,
    The pharmaceutical composition according to claim 9, comprising:
  11. 　ＣＤ３に結合する抗体又はその抗原結合断片が、
    配列番号４６で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号４６で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号４７で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号４７で表されるアミノ酸配列の１３５番目～２４１番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５１で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号５１で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号４８で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号４８で表されるアミノ酸配列の１３５番目～２４１番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号４９で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号４９で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５０で表されるアミノ酸配列の２番目～１１９番目のアミノ酸配列からなる重鎖可変領域及び配列番号５０で表されるアミノ酸配列の１３５番目～２４３番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５４で表されるアミノ酸配列の２７２番目～３８９番目のアミノ酸配列からなる重鎖可変領域及び配列番号５４で表されるアミノ酸配列の４０５番目～５１１番目のアミノ酸配列からなる軽鎖可変領域、
    配列番号５５で表されるアミノ酸配列の２７７番目～３９４番目のアミノ酸配列からなる重鎖可変領域及び配列番号５５で表されるアミノ酸配列の４１０番目～５１６番目のアミノ酸配列からなる軽鎖可変領域、又は、
    配列番号５６で表されるアミノ酸配列の２７７番目～３９４番目のアミノ酸配列からなる重鎖可変領域及び配列番号５６で表されるアミノ酸配列の４１０番目～５１６番目のアミノ酸配列からなる軽鎖可変領域、を含む、請求項８又は９に記載の医薬組成物。     An antibody that binds to CD3 or an antigen-binding fragment thereof,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48, and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49,
    A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50,
    A heavy chain variable region consisting of the 272nd to 389th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54, and a light chain variable region consisting of the 405th to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54,
    A heavy chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, and a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, Or
    A heavy chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56, The pharmaceutical composition according to claim 8 or 9, comprising:
  12. 　ＣＤ３に結合する抗体又はその抗原結合断片が、ｓｃＦｖである、請求項１～１１のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof that binds to CD3 is an scFv.
  13. 　ＣＤ３に結合する抗体又はその抗原結合断片が、
    配列番号４６で表されるアミノ酸配列の２番目～２４３番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号４７で表されるアミノ酸配列の２番目～２４１番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号５１で表されるアミノ酸配列の２番目～２４３番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号４８で表されるアミノ酸配列の２番目～２４１番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号４９で表されるアミノ酸配列の２番目～２４３番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号５０で表されるアミノ酸配列の２番目～２４３番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号５４で表されるアミノ酸配列の２７２番目～５１１番目のアミノ酸配列からなるｓｃＦｖ、
    配列番号５５で表されるアミノ酸配列の２７７番目～５１６番目のアミノ酸配列からなるｓｃＦｖ、または
    配列番号５６で表されるアミノ酸配列の２７７番目～５１６番目のアミノ酸配列からなるｓｃＦｖである、請求項１～１２のいずれか一つに記載の医薬組成物。     An antibody that binds to CD3 or an antigen-binding fragment thereof,
    scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46,
    scFv consisting of the 2nd to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 47,
    scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51,
    scFv consisting of the 2nd to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 48,
    scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49,
    scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50,
    scFv consisting of the 272nd to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54,
    Claim 1: An scFv consisting of the 277th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55, or an scFv consisting of the 277th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56. The pharmaceutical composition according to any one of items 1 to 12.
  14. 　Ｎ末端からＣ末端に向かって、ｓｃＦｖであるＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片、ｓｃＦｖであるＣＤ３に結合する抗体又はその抗原結合断片及びＦｃ領域（ｉ）をその順に含んでなる第１のポリペプチド；並びに、Ｆｃ領域（ｉｉ）を含む第２ポリペプチドを含み、好適には、Ｆｃ領域（ｉ）とＦｃ領域（ｉｉ）において会合してなる、請求項１～１３のいずれか一つに記載の医薬組成物。 In order from the N-terminus to the C-terminus, an antibody that binds to HLA/NY-ESO that is an scFv or an antigen-binding fragment thereof, an antibody that binds to CD3 that is an scFv or an antigen-binding fragment thereof, and an Fc region (i). and a second polypeptide comprising an Fc region (ii), preferably associated at the Fc region (i) and the Fc region (ii). A pharmaceutical composition according to any one of the following.
  15. 　第１ポリペプチドが、配列番号３２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列を含む、請求項１４に記載の医薬組成物。 The first polypeptide is represented by the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35. The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37. , the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40. The amino acid sequence of the 21st to 511th amino acids of the amino acid sequence represented by SEQ ID NO: 41, the amino acid sequence of the 21st to 511th of the amino acid sequence represented by SEQ ID NO: 42, the amino acid sequence of the 21st to 511th amino acids of the amino acid sequence represented by SEQ ID NO: 42, The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, Amino acid sequence, amino acid sequence from 21st to 511th of the amino acid sequence represented by SEQ ID NO: 53, amino acid sequence from 21st to 511th of the amino acid sequence represented by SEQ ID NO: 54, amino acid sequence represented by SEQ ID NO: 55 The pharmaceutical composition according to claim 14, comprising the 21st to 516th amino acid sequence of or the 21st to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56.
  16. 　第１ポリペプチドが、配列番号３２、３４、３５、３６、３７、３８、３９、４０、４１、４２、４３、３３、５２、５３もしくは５４で表されるアミノ酸配列の５２９番目～７４５番目のアミノ酸配列、又は、配列番号５５もしくは５６で表されるアミノ酸配列の５３４番目～７５０番目のアミノ酸配列を含む、請求項１４又は１５に記載の医薬組成物。 The first polypeptide is located at positions 529 to 745 of the amino acid sequence represented by SEQ ID NO: 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 33, 52, 53 or The pharmaceutical composition according to claim 14 or 15, comprising the amino acid sequence or the 534th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 55 or 56.
  17. 　第１ポリペプチドが、配列番号３２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、列番号５５で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列からなる、請求項１４～１６のいずれか一つに記載の医薬組成物。 The first polypeptide is represented by the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35. The 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37. , the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40. Amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: 41, amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: 42, amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: The 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, Amino acid sequence, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence represented by SEQ ID NO: 55 The pharmaceutical composition according to any one of claims 14 to 16, consisting of the 20th to 750th amino acid sequence of or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56.
  18. 　第２ポリペプチドが、配列番号３１で示されるアミノ酸配列の２０番目～２４６番目のアミノ酸配列を含んでなる、請求項１４～１７のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 14 to 17, wherein the second polypeptide comprises the 20th to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 31.
  19. 　第１ポリペプチド、第２ポリペプチド及び、第３ポリペプチドを含み、
    第１ポリペプチドが、Ｎ末端からＣ末端に向かって、ｓｃＦｖであるＨＬＡ／ＮＹ－ＥＳＯに結合する抗体又はその抗原結合断片、ｓｃＦｖであるＣＤ３に特異的に結合する抗体又はその抗原結合断片及びＦｃ領域（ｉ）を、その順に含んでなり、
    　第２ポリペプチドが、Ｆｃ領域（ｉｉ）を含む免疫グロブリン重鎖からなり、
    　第３ポリペプチドが免疫グロブリン軽鎖からなり、
    　好適には、第２ポリペプチドと第３ポリペプチドが会合しており、
    　第１ポリペプチドと第２ペプチドが、それぞれのＦｃ領域において会合している、請求項１～１３のいずれか一つに記載の医薬組成物。     comprising a first polypeptide, a second polypeptide, and a third polypeptide,
    The first polypeptide includes, from the N-terminus to the C-terminus, an antibody or antigen-binding fragment thereof that binds to HLA/NY-ESO, which is an scFv, an antibody or antigen-binding fragment thereof, which specifically binds to CD3, which is an scFv, and Fc region (i) in that order,
    the second polypeptide consists of an immunoglobulin heavy chain comprising an Fc region (ii);
    the third polypeptide consists of an immunoglobulin light chain,
    Preferably, the second polypeptide and the third polypeptide are associated,
    The pharmaceutical composition according to any one of claims 1 to 13, wherein the first polypeptide and the second peptide are associated in their respective Fc regions.
  20. 　第２ポリペプチドが、配列番号４４で表されるアミノ酸配列のアミノ酸番号２０～２４２のアミノ酸配列を含む、請求項１９に記載の医薬組成物。 The pharmaceutical composition according to claim 19, wherein the second polypeptide comprises the amino acid sequence of amino acids 20 to 242 of the amino acid sequence represented by SEQ ID NO: 44.
  21. 　第３ポリペプチドが配列番号４５で表されるアミノ酸配列を含む、請求項１９又は２０に記載の医薬組成物。 The pharmaceutical composition according to claim 19 or 20, wherein the third polypeptide comprises the amino acid sequence represented by SEQ ID NO: 45.
  22. 　第１ポリペプチドが、配列番号３２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号３３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２１番目～５１１番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２１番目～５１６番目のアミノ酸配列を含む、請求項１９～２１のいずれか一つに記載の医薬組成物。 The first polypeptide is represented by the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35. The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37. , the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40. The amino acid sequence of the 21st to 511th amino acids of the amino acid sequence represented by SEQ ID NO: 41, the amino acid sequence of the 21st to 511th of the amino acid sequence represented by SEQ ID NO: 42, the amino acid sequence of the 21st to 511th amino acids of the amino acid sequence represented by SEQ ID NO: 42, The 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, the 21st to 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, Amino acid sequence, amino acid sequence from 21st to 511th of the amino acid sequence represented by SEQ ID NO: 53, amino acid sequence from 21st to 511th of the amino acid sequence represented by SEQ ID NO: 54, amino acid sequence represented by SEQ ID NO: 55 The pharmaceutical composition according to any one of claims 19 to 21, comprising the 21st to 516th amino acid sequence of or the 21st to 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56.
  23. 　第１ポリペプチドが、配列番号３２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３５で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３６で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３７で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３８で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号３９で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４０で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４１で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号４３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列及び配列番号３３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５２で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５３で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５４で表されるアミノ酸配列の２０番目～７４５番目のアミノ酸配列、配列番号５５で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列、又は、配列番号５６で表されるアミノ酸配列の２０番目～７５０番目のアミノ酸配列からなる、請求項１９～２２のいずれか一つに記載の医薬組成物。 The first polypeptide is represented by the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 32, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35. The 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 36, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 37. , the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 38, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 39, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40. Amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: 41, amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: 42, amino acid sequence from 20th to 745th of the amino acid sequence represented by SEQ ID NO: The 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 33, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52. Amino acid sequence, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence represented by SEQ ID NO: 55 The pharmaceutical composition according to any one of claims 19 to 22, consisting of the 20th to 750th amino acid sequence of or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 56.
  24. 　Ｎ末端からＣ末端に向かって、
    ｓｖＦｖであるＨＬＡ／ＮＹ－ＥＳＯに特異的に結合する抗体又はその抗原結合断片、ＣＤ３に結合する抗体の重鎖可変領域及び定常領域ＣＨ１並びに免疫グロブリンＦｃ領域（ｉ）をその順で含んでなる第１ポリペプチド、
    免疫グロブリンのヒンジ領域及びＦｃ領域（ｉｉ）を含んでなる第２ポリペプチド、並びに、
    可変領域及び定常領域からなるＣＤ３に結合する抗体軽鎖を含む第３ポリペプチドを含み、
    好適には、第１ポリペプチドと第２ポリペプチドがＦｃ領域（ｉ）とＦｃ領域（ｉｉ）において会合しており、第１ポリペプチドが抗体重鎖の可変領域及び定常領域ＣＨ１において第３ポリペプチドと会合してなる、請求項９～１１のいずれか一つに記載の医薬組成物。     From the N-terminus to the C-terminus,
    An antibody that specifically binds to HLA/NY-ESO, which is svFv, or an antigen-binding fragment thereof, comprising the heavy chain variable region and constant region CH1 of an antibody that binds to CD3, and immunoglobulin Fc region (i) in that order. a first polypeptide,
    a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii); and
    comprising a third polypeptide comprising an antibody light chain that binds to CD3, consisting of a variable region and a constant region,
    Preferably, the first polypeptide and the second polypeptide are associated in Fc region (i) and Fc region (ii), and the first polypeptide is associated with a third polypeptide in the variable region and constant region CH1 of the antibody heavy chain. The pharmaceutical composition according to any one of claims 9 to 11, which is associated with a peptide.
  25. 　第１ポリペプチドが、配列番号５７で表されるアミノ酸配列の２０番目～７２４番目のアミノ酸配列、配列番号６０で表されるアミノ酸配列の２０番目～７１９番目のアミノ酸配、又は、配列番号６１で表されるアミノ酸配列２０番目～７１９番目のアミノ酸配列を含む、請求項９～１１及び２４のいずれか一つに記載の医薬組成物。 The first polypeptide is the 20th to 724th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 57, the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 60, or the amino acid sequence of SEQ ID NO: 61. 25. The pharmaceutical composition according to any one of claims 9 to 11 and 24, comprising the amino acid sequence of positions 20 to 719 of the expressed amino acid sequence.
  26. 　第２ポリペプチドが、配列番号３１で示されるアミノ酸配列の２０番目～２４６番目のアミノ酸配列を含んでなる、請求項９～１１、２４及び２５のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 9 to 11, 24, and 25, wherein the second polypeptide comprises the 20th to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 31.
  27. 　第３ポリペプチドが、配列番号５８で表されるアミノ酸配列の２１番目～１２７番目のアミノ酸配列を含んでなる、請求項９～１１及び２４～２６のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 9 to 11 and 24 to 26, wherein the third polypeptide comprises the 21st to 127th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 58.
  28. 　第３ポリペプチドが、配列番号５８で表されるアミノ酸配列の２１番目～２３３番目のアミノ酸配列を含んでなる、請求項９～１１及び２４～２７のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 9 to 11 and 24 to 27, wherein the third polypeptide comprises the 21st to 233rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 58.
  29. 　請求項１～２８のいずれか一つに記載の医薬組成物であって、該医薬組成物に含まれる１つ又は２つ以上のポリペプチドの有するアミノ酸配列において、該配列のカルボキシル末端から１つ又は２つのアミノ酸が欠失してなる、該医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 28, wherein in the amino acid sequence of one or more polypeptides contained in the pharmaceutical composition, one amino acid sequence from the carboxyl terminus of the sequence is Or the pharmaceutical composition, wherein two amino acids are deleted.
  30. 　がんが、腎がん、メラノーマ、有棘細胞がん、基底細胞がん、結膜がん、口腔がん、喉頭がん、咽頭がん、甲状腺がん、肺がん（非小細胞肺がん（腺がん、扁平上皮がん、大細胞がん）、小細胞肺がん）、乳がん、食道がん、胃がん、十二指腸がん、小腸がん、大腸がん、直腸がん、虫垂がん、肛門がん、肝がん、胆嚢がん、胆管がん、膵がん、副腎がん、膀胱がん、前立腺がん、子宮がん、膣がん、脂肪肉腫、血管肉腫、軟骨肉腫、横紋筋肉腫、ユーイング肉腫、骨肉腫、未分化多型肉腫、粘液型線維肉腫、悪性末梢性神経鞘腫、後腹膜肉腫、滑膜肉腫、子宮肉腫、消化管間質腫瘍、平滑筋肉腫、類上皮肉腫、Ｂ細胞リンパ腫、Ｔ・ＮＫ細胞リンパ腫、ホジキンリンパ腫、骨髄性白血病、リンパ性白血病、骨髄増殖性疾患、骨髄異形成症候群、多発性骨髄腫、精巣がん及び卵巣がんからなる群から選択される１つ又は２つ以上である、請求項１～２９のいずれか一つに記載の医薬組成物。 Cancer: renal cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cavity cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer (non-small cell lung cancer) cancer, squamous cell carcinoma, large cell carcinoma), small cell lung cancer), breast cancer, esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, colon cancer, rectal cancer, appendiceal cancer, anal cancer, Liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal gland cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, B 1 selected from the group consisting of cell lymphoma, T/NK cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, testicular cancer, and ovarian cancer. The pharmaceutical composition according to any one of claims 1 to 29, which is one or more.
  31. 　免疫チェックポイント分子を阻害する化合物又は化学療法剤の後に投与される、請求項１～３０のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 30, which is administered after a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule.
  32. 　免疫チェックポイント分子を阻害する化合物又は化学療法剤より前に投与される、請求項１～３０のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 30, which is administered before a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule.
  33. 　免疫チェックポイント分子を阻害する化合物又は化学療法剤と同時に投与される、請求項１～３０のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 30, which is administered simultaneously with a compound or chemotherapeutic agent that inhibits immune checkpoint molecules.
  34. 　免疫チェックポイント分子を阻害する化合物又は化学療法剤を含む、請求項１～３０及び３３のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 30 and 33, comprising a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule.
  35. 　さらなる１つ又は２つ以上の医薬及び／又は治療と組み合わせて使用される、請求項１～３４のいずれか一つに記載の医薬組成物。 Pharmaceutical composition according to any one of claims 1 to 34, used in combination with one or more further medicaments and/or treatments.
  36. 　［ｉｉ］記載の化合物が免疫チェックポイント分子を阻害する化合物である、請求項１～３５のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 35, wherein the compound described in [ii] is a compound that inhibits an immune checkpoint molecule.
  37. 　免疫チェックポイント分子がＰＤ－１、ＰＤ－Ｌ１、ＰＤ－Ｌ２、ＣＴＬＡ－４及びＴＩＧＩＴよりなる群から選択される、請求項１～３６のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 36, wherein the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4 and TIGIT.
  38. 　免疫チェックポイント分子がＰＤ－１である、請求項１～３７のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 37, wherein the immune checkpoint molecule is PD-1.
  39. 　免疫チェックポイント分子を阻害する化合物がニボルマブ若しくはペムブロリズマブ、その抗原結合断片、又は、その抗原結合断片を含む化合物である、請求項３８記載の医薬組成物。 39. The pharmaceutical composition according to claim 38, wherein the compound that inhibits an immune checkpoint molecule is nivolumab or pembrolizumab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof.
  40. 　免疫チェックポイント分子がＰＤ－Ｌ１である、請求項１～３７のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 37, wherein the immune checkpoint molecule is PD-L1.
  41. 　免疫チェックポイント分子を阻害する化合物がアテゾリズマブ、デュルバルマブ若しくはアベルマブ、その抗原結合断片、又は、その抗原結合断片を含む化合物である、請求項４０記載の医薬組成物。 41. The pharmaceutical composition according to claim 40, wherein the compound that inhibits an immune checkpoint molecule is atezolizumab, durvalumab, or avelumab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof.
  42. 　免疫チェックポイント分子がＣＴＬＡ－４である、請求項１～３７のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 37, wherein the immune checkpoint molecule is CTLA-4.
  43. 　免疫チェックポイント分子を阻害する化合物がイピリムマブ、トレメリムマブ、スパルタリズマブ若しくはセミプリマブ、その抗原結合断片、又は、その抗原結合断片を含む化合物である、請求項４２記載の医薬組成物。 43. The pharmaceutical composition according to claim 42, wherein the compound that inhibits an immune checkpoint molecule is ipilimumab, tremelimumab, spartalizumab, or cemiplimab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof.
  44. 　免疫チェックポイント分子がＴＩＧＩＴである、請求項１～３７のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 37, wherein the immune checkpoint molecule is TIGIT.
  45. 　免疫チェックポイント分子を阻害する化合物がチラゴルマブ若しくはビボストリマブ、その抗原結合断片、又は、その抗原結合断片を含む化合物である、請求項４４記載の医薬組成物。 45. The pharmaceutical composition according to claim 44, wherein the compound that inhibits an immune checkpoint molecule is tiragolumab or vivostrimab, an antigen-binding fragment thereof, or a compound containing an antigen-binding fragment thereof.
  46. 　免疫チェックポイント分子を阻害する化合物がペムブロリズマブ、ニボルマブ、スパルタリズマブ、セミプリマブ、アベルマブ、アテゾリズマブ、ヂュルバルマブ、イピリムマブ、又は、トレメリムマブである、請求項１～３６のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 36, wherein the compound that inhibits an immune checkpoint molecule is pembrolizumab, nivolumab, spartalizumab, cemiplimab, avelumab, atezolizumab, durvalumab, ipilimumab, or tremelimumab.
  47. 　免疫チェックポイント分子を阻害する化合物が、ペムブロリズマブ若しくはペムブロリズマブの抗原結合断片、又は、その抗原結合断片を含む化合物である、請求項１～３９のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 39, wherein the compound that inhibits an immune checkpoint molecule is pembrolizumab, an antigen-binding fragment of pembrolizumab, or a compound containing an antigen-binding fragment thereof.
  48. 　［ｉｉ］記載の化合物が化学療法剤である、請求項１～３５のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 35, wherein the compound according to [ii] is a chemotherapeutic agent.
  49. 　化学療法剤が微小管形成阻害剤である、請求項１～３５及び４８のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 35 and 48, wherein the chemotherapeutic agent is a microtubule formation inhibitor.
  50. 　化学療法剤がタキサン系の微小管形成阻害剤である、請求項１～３５、４８及び４９のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 35, 48, and 49, wherein the chemotherapeutic agent is a taxane-based microtubule formation inhibitor.
  51. 　化学療法剤がパクリタキセル、ドセタキセル、ビンクリスチン、ビンブラスチン、ビンデシン、エリブリン、ビノレルビン、アルブミン懸濁型パクリタキセルオキサリプラチン、カルボプラチン、シスプラチン、ネダプラチン、アザシチジン、デシタビン、ドキソルビシン、ブレオマイシン、リポソーマルドキソルビシン、イホスファミド、シクロホスファミド、及び、ダカルバジンよりなる群から選択される、請求項１～３５及び４８～５０のいずれか一つに記載の医薬組成物。 Chemotherapy agents include paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, albumin suspension paclitaxel oxaliplatin, carboplatin, cisplatin, nedaplatin, azacitidine, decitabine, doxorubicin, bleomycin, liposomal doxorubicin, ifosfamide, cyclophosphamide The pharmaceutical composition according to any one of claims 1 to 35 and 48 to 50, selected from the group consisting of dacarbazine and dacarbazine.
  52. 　化学療法剤がパクリタキセル又はアルブミン懸濁型パクリタキセルである、請求項１～３５及び４８～５１のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 35 and 48 to 51, wherein the chemotherapeutic agent is paclitaxel or albumin-suspended paclitaxel.
  53. 　さらにマルチキナーゼ阻害剤と組み合わせて使用される、請求項１～５２のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 52, further used in combination with a multikinase inhibitor.
  54. 　マルチキナーゼ阻害剤がソラフェニブ、スニチニブ、パゾパニブ、レゴラフェニブ、及びレンバチニブよりなる群から選択される１つ又は２つ以上である、請求項５３に記載の医薬組成物。 54. The pharmaceutical composition according to claim 53, wherein the multikinase inhibitor is one or more selected from the group consisting of sorafenib, sunitinib, pazopanib, regorafenib, and lenvatinib.
  55. 　マルチキナーゼ阻害剤が、レンバチニブである、請求項５３又は５４記載の医薬組成物。 The pharmaceutical composition according to claim 53 or 54, wherein the multikinase inhibitor is lenvatinib.
  56. 　［ｉｉ］記載の化合物が、免疫チェックポイント分子を阻害する化合物であり、好適には、ペムブロリズマブ若しくはペムブロリズマブの抗原結合断片、又はその抗原結合断片を含む化合物である、請求項５３～５５のいずれか一つに記載の医薬組成物。 Any one of claims 53 to 55, wherein the compound described in [ii] is a compound that inhibits an immune checkpoint molecule, and is preferably a compound comprising pembrolizumab, an antigen-binding fragment of pembrolizumab, or an antigen-binding fragment thereof. Pharmaceutical composition according to one of the above.
  57. 　免疫チェックポイント分子を阻害する化合物が、ペムブロリズマブ若しくはペムブロリズマブの抗原結合断片、又はその抗原結合断片を含む化合物であり、マルチキナーゼ阻害剤が、レンバチニブである、請求項５３～５６のいずれか一つに記載の医薬組成物。 57. The compound that inhibits an immune checkpoint molecule is pembrolizumab, an antigen-binding fragment of pembrolizumab, or a compound comprising an antigen-binding fragment thereof, and the multikinase inhibitor is lenvatinib. Pharmaceutical compositions as described.
  58. 　化学療法剤がパクリタキセル、ドセタキセル、ビンクリスチン、ビンブラスチン、ビンデシン、エリブリン、ビノレルビン及びアルブミン懸濁型パクリタキセルよりなる群から選択されるタキサン系の微小管形成阻害剤である、請求項１～３５及び４８～５２のいずれか一つに記載の医薬組成物。 Claims 1 to 35 and 48 to 52, wherein the chemotherapeutic agent is a taxane microtubule formation inhibitor selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, and albumin-suspended paclitaxel. A pharmaceutical composition according to any one of the following.
  59. 　がんの治療及び／又は予防のための、請求項１～４７のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤。 A compound or chemotherapeutic agent that inhibits [i] the multispecific antibody and [ii] immune checkpoint molecule according to any one of claims 1 to 47 for the treatment and/or prevention of cancer.
  60. 　がんの治療及び／又は予防のための、請求項５３～５７のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤、並びに、マルチキナーゼ阻害剤。 [i] the multispecific antibody and [ii] a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule according to any one of claims 53 to 57 for the treatment and/or prevention of cancer, and , a multikinase inhibitor.
  61. 　がんの治療及び／又は予防のための、請求項１～４７のいずれか一つに記載［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤。 A compound or chemotherapeutic agent that inhibits [i] a multispecific antibody and [ii] an immune checkpoint molecule according to any one of claims 1 to 47 for the treatment and/or prevention of cancer.
  62. 　がんの治療及び／又は予防のための医薬組成物を調製するための、請求項５３～５７のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤、並びに、マルチキナーゼ阻害剤の使用。 [i] multispecific antibody and [ii] inhibiting immune checkpoint molecules according to any one of claims 53 to 57 for preparing a pharmaceutical composition for the treatment and/or prevention of cancer. compounds or chemotherapeutic agents, as well as the use of multikinase inhibitors.
  63. 　請求項１～４７のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤を投与することを含む、がんを治療及び／又は予防する方法。 Treating and/or How to prevent it.
  64. 　請求項５３～５７のいずれか一つに記載の［ｉ］多重特異性抗体及び[ｉｉ] 免疫チェックポイント分子を阻害する化合物又は化学療法剤、並びに、マルチキナーゼ阻害剤を投与することを含む、がんを治療及び／又は予防する方法。 comprising administering [i] a multispecific antibody and [ii] a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule, and a multikinase inhibitor according to any one of claims 53 to 57. Methods of treating and/or preventing cancer.
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Publication number Priority date Publication date Assignee Title
WO2021200857A1 (en) * 2020-03-30 2021-10-07 国立大学法人三重大学 Bispecific antibody
JP2021528423A (en) * 2018-06-21 2021-10-21 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． How to treat cancer with bispecific anti-CD3 × MUC16 antibody and anti-PD-1 antibody

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021528423A (en) * 2018-06-21 2021-10-21 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． How to treat cancer with bispecific anti-CD3 × MUC16 antibody and anti-PD-1 antibody
WO2021200857A1 (en) * 2020-03-30 2021-10-07 国立大学法人三重大学 Bispecific antibody

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